Combinatorial gamma 9 delta 2 t cell receptor chain exchange

ABSTRACT

The current invention provides methods to identify γ9δ2T-cell receptors (γ9δ2TCR) that mediate anti-tumour responses. Surprisingly, it was now found that the CDR3 regions of the γ9-T-cell receptor chain and the δ2-T-Cell receptor chain (δ2TCR chain) are of importance. Based on these findings, combinatorial-γδTCR-chain-exchange (CTE) is proposed as an efficient method for identifying γ9δ2TCRs that mediate anti-tumour responses. Using the method of the invention, specific sequences of the respective γ9TCR and δ2TCR chains were identified that mediate anti-tumour responses. Hence, the invention further provides for specific γ9δ2TCRs, or fragments thereof, that may be used e.g. in diagnostics or treatment of cancer. The invention further provides for nucleic acid sequences, genetic constructs and retroviral vectors that can be used to express the γ9δ2TCRs according to the invention.

FIELD OF THE INVENTION

The invention is in the field of medicine. It relates to immunology andto cell therapy for the treatment of cancer. The invention furtherrelates to the methods for identification of T cell receptors, inparticular T cell receptors that may mediate anti-tumour responses. Theinvention further relates to specific T-cell receptors that mediateanti-tumour responses and uses thereof, in the treatment of cancer.

BACKGROUND ART

So far, allogeneic stem cell transplantation (allo-SCT) is the only wellestablished and proven curative cellular therapy for patients sufferingfrom cancer. This is demonstrated by the observation that long termremissions can be in particular achieved with allo-SCT in patients with“poor-risk” leukaemia. However, cure comes at a price of severetoxicity, resulting in an overall mortality of approximately 30% oftreated patients. This hampers broad clinical implementation as inparticular patients with low risk cancers might have a good overallsurvival not justifying such aggressive therapies. The ultimate goalremains therefore the development of a non-aggressive transplantationmethod. This means the generation of cellular products equipped withmolecular features that allow selective targeting of the cancer cellwhile preserving healthy tissues. This would allow a curative treatmentto a large number of patients with medical need, irrespective of age.

To engineer a transplant, which may be allogeneic or autologous, tofacilitate the rapid generation of e.g. tumour-reactive αβT-cells, ithas been proposed to reprogram αβT-cells with genes encoding for atumour-specific αβT-cell receptor (TCR) or a chimeric antigen receptor.Several of such receptors are already being used to redirect αβT-cellsin phase I clinical trials. However, reprogramming αβT-cells withdefined αβTCRs is substantially hampered by their restriction to HLAtypes, limiting thereby the number of patients that can be treated withone αβ-TCR. In addition, pairing of introduced αβTCR chains withendogenous αβTCR chains can induce life-threatening auto-reactivity.

It has been proposed to mediate a selective anti-tumour-reactivity witha high-affinity TCR using the ability of γ9δ2T-cells to mediateanti-tumour-reactivity while ignoring healthy-environment (Fisch et al.,1990, Science 250, 1269-1273). Isolated γ9δ2T-cells may efficiently killtumour-cells of haematological malignancies and solid tumours (Kabelitzet al., 2007, Cancer Res. 67, 5-8). However, the function andproliferation capacity of γ9δ2T-cells is frequently heavily impaired incancer patients making autologous γ9δ2T-cells less attractive for immuneinterventions.

It has been proposed to transfer a defined γ9δ2TCR into αβT-cells, whichmediates a tumour-specific proliferation of αβT-cells and redirects botheffector CD8+ and helper CD4+ αβT-cell subsets against a broad panel oftumour-cell-lines while normal cells in vitro and in vivo may be ignored(Marcu-Malina et al., 2011, Blood 118, 50-59). However, little knowledgeis available about how γ9δ2TCRs mediate different activity againsttumour cells and what strategy needs to be used to isolate γ9δ2TCRs withhigh activity against cancer cells.

Hence, there is need in the art to provide for γ9δ2TCRs with highactivity against cancer cells and there is a need in the art to providefor improved methods for selecting such highly active γ9δ2TCRs.

SUMMARY OF THE INVENTION

The current invention provides methods to identify γ9δ2T-cell receptors(γ9δ2TCR) that mediate anti-tumour responses. As said, little knowledgeis available about the molecular requirements of a γ9δ2TCRs to mediateanti-tumour reactivity. Especially the contribution of the variabilitywithin the complementary determining regions (CDRs) of the γ9δ2TCR formediating anti-tumour responses was regarded as negligible in the priorart. Surprisingly, it was now found that the CDR3 regions of theγ9T-cell receptor chain (γ9TCR chain) and the δ2T-cell receptor chain(δ2TCR chain) are of importance. Based on these findings,combinatorial-γδTCR-chain-exchange (CTE) is proposed as an efficientmethod for identifying γ9δ2TCRs that mediate anti-tumour responses. Inthe methods of the invention, the CDR3 regions of the γ9T-cell receptorchain and the δ2T-cell receptor chain are randomly modified and combinedto form a novel γ9δ2TCR. The newly designed γ9δ2TCR is provided andpreferably integrated into T-cells, which subsequently express a γ9δ2TCRat the cell surface. Accordingly the anti-tumour response of theCTE-engineered T-cells is determined. In this way multiple combinationscan be tested, and for each combination the anti-tumour response can bedetermined. After determining the anti-tumour response, γ9δ2T-cellreceptors that mediate highly active anti-tumour responses can beidentified. Using the method of the invention, such as described in theexamples, already several responsive γ9δ2TCRs were identified thatmediate increased anti-tumour responses compared to the referenceγ9δ2TCR G115wt. Hence, the invention further provides for γ9δ2TCRs, orfragments thereof, that may be used e.g. in diagnostics or the treatmentof cancer. The invention further provides for nucleic acid sequences,genetic constructs and retroviral vectors that can be used to expressthe γ9δ2TCRs in cells, preferably T cells, according to the invention.

FIGURES

FIG. 1: Anti-tumour reactivity mediated by γ9δ2TCRs.

Peripheral blood T-cells were virally transduced with indicated wildtypeγ9δ2TCRs or CTE-engineered γ9δ2TCRs and tested against Daudi (A, C) in a⁵¹Cr-release assay (E:T 3:1). Specific lysis is indicated as fold change⁵¹Cr-release measured in the supernatant after 5 h. Fold change wascalculated when compared to reactivity of γ9-G115_(wt)/δ2-G115_(wt)engineered T-cells. (B, D) in an IFNγ ELISA in the presence of indicatedamounts of pamidronate or (E) different E:T ratios. (F) Percentages ofcell-cell conjugates of Daudi and T-cells engineered with indicatedγ9δ2TCR were determined by flow cytometry. Data represent the mean±SD.*p<0.05, **p<0.01, ***p<0.001 by 1-way ANOVA.

FIG. 2: γ9δ2TCR expression and functional avidity of transduced T-cellsexpressing single alanine mutated δ2 chain of clone G115.

Peripheral blood T-cells were virally transduced with indicated γ9 andδ2 TCR chains and (A) analyzed by flow cytometry using a δ2-chainspecific antibody. Shown is the fold change in mean fluorescentintensity (MFI) in comparison to wildtype control expressingδ2-G115_(wt). (C) Lytic activity of transductants was tested in a⁵¹Cr-release assay against the tumour target Daudi (E:T 10:1). Specificlysis is indicated as fold change ⁵¹Cr-release measured in thesupernatant after 5 h. Fold change was calculated as compared toreactivity of unmutated wildtype (δ2-G115_(wt)). Arrows indicatemutations in δ2-G115 that impaired receptor expression (dashed arrows)or functional avidity (solid arrows). (B, D) Crystal structure ofγ9δ2TCR G115 indicating relevant amino acids (arrows).

FIG. 3: γ9δ2TCR expression and functional avidity of transduced T-cellsexpressing γ9δ2TCR G115 with δ2-CDR3 length mutations.

(A) γ9δ2TCR expression of indicated transductants was analyzed by flowcytometry using a γδTCR-pan antibody. Shown is the fold change in meanfluorescent intensity (MFI) in comparison to wildtype control expressingδ2-G115_(wt). (B) IFNγ secretion of δ2-G115_(LM) transduced T-cellsagainst the tumour target Daudi (E:T 1:1) was measured by ELISA after 24h incubation in the presence of 100 μM pamidronate. Shown is the foldchange in IFNγ production when compared to reactivity of transductantsexpressing wt δ2-G115_(wt). (C) Transductants expressingδ2-G115_(LM0,1,4,12) were tested in a titration assay against the tumourtarget Daudi with increasing amounts of pamidronate as indicated. IFNγproduction was measured after 24 h by ELISA. (D) Generated δ2-G115_(LMs)were matched in a BLAST search with γ9δ2TCRs described in the IMGTdatabase. Shown is the number of citations compared to δ2-G115_(LM) ofsimilar δCDR3 length. (E) Transductants with δ2-G115_(LM2,4,6) werecompared side-by-side to transductants expressing individual γ9δ2TCRs ofthe same SCDR3 length. IFNγ secretion of transduced T-cells against thetumour target Daudi (E:T 1:1) was measured by ELISA after 24 h in thepresence of 100 μM pamidronate. Shown is the fold change in IFNγproduction compared to reactivity of transductants expressing wtδ2-G115_(wt). Data represent the mean±SD. ^(**)p<0.01, ^(**)p<0.001 by1-way ANOVA. (F) Crystal structure of γ9δ2TCR G115; the region that wasused for alanine stretches within SCDR3 is shown in white, residualSCDR3 in green, δ chain in blue, γ chain in brown.

FIG. 4: Functional avidity of transduced T-cells expressing γ9δ2TCR G115with γ9-CDR3 length mutations.

(A) Peripheral blood T-cells were virally transduced with indicated γ9and δ2 TCR chains. Lytic activity of transductants was comparedside-by-side to T-cells expressing individual γ9δ2TCRs of the sameγ9CDR3 length. Specific lysis is indicated as fold change ⁵¹Cr-releasemeasured in the supernatant after 5 h. Data represent the mean±SD.^(**)p<0.01 by 1-way ANOVA. (B) Crystal structure of γ9δ2TCR G115indicating γ9CDR3 in gray including amino acids γ9-G115_(A109),γ9-G115_(Q110) and γ9-G115_(Q111) (red arrows), SCDR3 is shown in green;δ chain in blue; γ chain in brown.

FIG. 5: Anti-tumour reactivity of T-cells transduced with CTE-engineeredγ9δ2TCRs in vitro.

Peripheral blood T-cells were virally transduced with indicated γ9δ2TCRsand tested against indicated tumour cell lines and healthy controltissue. (A) Transductants were incubated with target cells (E:T 1:1) inthe presence of 10 μM pamidronate. IFNγ production was measured after 24h by ELISA. Data represent the mean±SD. *p<0.05, ^(**)p<0.01,^(***)p<0.001 by 1-way ANOVA. (B) Transductants were incubated withindicated tumour targets loaded with ⁵¹Cr (E:T 10:1). Percentage ofspecific lysis was determined by ⁵¹Cr-release measured in thesupernatant after 5 h. (C) CTE-engineered T-cells were tested againstprimary AML blasts and healthy progenitor cells in an IFNγ ELISpot assay(E:T 3:1) in the presence of 10 μM pamidronate. Data represent themean±SD. *p<0.05, ^(**)p<0.01, ^(***)p<0.001 by 1-way ANOVA.

FIG. 6: Anti-tumour reactivity of T-cells transduced with CTE-engineeredγ9δ2TCRs in vivo.

The functional avidity of T-cells expressing CTE-γ9δ2TCRγ9-G115_(wt)/δ2-cl5_(wt) or control γ9δ2TCR (γ9-G115_(wt)/δ2-G115_(wt))was studied in Rag2^(−/−)yc^(−/−) double knockout mice (4-7 mice pergroup). After total body irradiation (2Gy) on day 0, mice wereintravenously injected with 0.5×10⁶ Daudi-luciferase or 5×10⁶RPMI8226/S-luciferase cells and 10⁷ CTE-γ9δ2TCR transduced T-cells atday 1. Additionally, 6×10⁵ IU IL2 in IFA and pamidronate (10 mg/kg bodyweight) were injected at day 1 and every 3 weeks until the end of theexperiment. (A,B) Tumour outgrowth was assessed in vivo bybioluminescence imaging (BLI) by measuring the entire area of mice onboth sides. Data represent the mean of all animals measured (Daudi: n=4,RPMI8226/S: n=7). *p<0.05, ^(**)p<0.01 by 1-way ANOVA (Daudi: day 42;RPMI8226/S: day 35). (C) Overall survival of treated Daudi mice wasmonitored for 72 days. *p<0.05, p<0.01 by logrank (Mantel-Cox) test.

FIG. 7. Combinatorial-γδTCR-chain-exchange (CTE)

Individual γ9δ2T-cell clones are isolated (A) and the sequence of theγ9TCR and δ2TCR chain is determined (B). By Combinatorial-γδTCR-chainexchange novel γ9δ2TCRs are designed whereby γ9- and δ2CDR3 regions arerandomly modified (e.g. via amino acid substitutions, deletions and/orinsertions and/or shortening or stretching of the CDR3 length) (C) andnewly combined (D). Subsequently novel γ9δ2TCRs are introduced intocells, preferably T-cells such as αβT-cells (e.g. via transduction usinga retroviral vector encoding the γ9TCR and δ2TCR chains) andCTE-engineered T-cells are provided (E). Finally, CTE-engineered T-cellsare tested against tumour cells in functional assays to determineγ9δ2TCRs that mediate high anti-tumour responses. Hence, γ9δ2TCRs withe.g. the highest anti-tumour response and/or lowest side effects can beselected (F).

DEFINITIONS

In the following description and examples a number of terms are used. Inorder to provide a clear and consistent understanding of thespecifications and claims, including the scope to be given to suchterms, the following definitions are provided. Unless otherwise definedherein, all technical and scientific terms used have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. The disclosures of all publications, patentapplications, patents and other references are incorporated herein intheir entirety by reference.

Methods of carrying out the conventional techniques used in methods ofthe invention will be evident to the skilled worker. The practice ofconventional techniques in molecular biology, biochemistry,computational chemistry, cell culture, recombinant DNA, bioinformatics,genomics, sequencing and related fields are well-known to those of skillin the art and are discussed, for example, in the following literaturereferences: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2ndEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.,1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley& Sons, New York, 1987 and periodic updates; and the series Methods inEnzymology, Academic Press, San Diego.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. It encompasses the verbs “consisting essentially of”as well as “consisting of”.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. For example, amethod for isolating “a” DNA molecule, as used above, includes isolatinga plurality of molecules (e.g. 10's, 100's, 1000's, 10's of thousands,100's of thousands, millions, or more molecules).

Aligning and alignment: With the term “aligning” and “alignment” ismeant the comparison of two or more nucleotide sequences based on thepresence of short or long stretches of identical or similar nucleotides.Several methods for alignment of nucleotide sequences are known in theart, as will be further explained below. With the term “aligning” and“alignment” is also meant the comparison of two or more amino acidsequences based on the presence of short or long stretches of identicalor similar amino acids. Several methods for alignment of amino acidsequences are known in the art, as will be further explained below.

“Expression of a gene” refers to the process wherein a DNA region, whichis operably linked to appropriate regulatory regions, particularly apromoter, is transcribed into an RNA, which is biologically active, i.e.which is capable of being translated into a biologically active proteinor peptide (or active peptide fragment) or which is active itself (e.g.in posttranscriptional gene silencing or RNAi). An active protein incertain embodiments refers to a protein being constitutively active. Thecoding sequence is preferably in sense-orientation and encodes adesired, biologically active protein or peptide, or an active peptidefragment.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter, or rather atranscription regulatory sequence, is operably linked to a codingsequence if it affects the transcription of the coding sequence.Operably linked means that the DNA sequences being linked are typicallycontiguous and, where necessary to join two or more protein encodingregions, contiguous and in reading frame.

The term “genetic construct” means a DNA sequence comprising a region(transcribed region), which is transcribed into an RNA molecule (e.g. anmRNA) in a cell, operably linked to suitable regulatory regions (e.g. apromoter). A genetic construct may thus comprise several operably linkedsequences, such as a promoter, a 5′ leader sequence comprising e.g.sequences involved in translation initiation, a (protein) encodingregion, splice donor and acceptor sites, intronic and exonic sequences,and a 3′ non-translated sequence (also known as 3′ untranslated sequenceor 3′UTR) comprising e.g. transcription termination sequence sites.

“Identity” is a measure of the identity of nucleotide sequences or aminoacid sequences. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. See, e.g.:(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS,Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OFSEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, vonHeinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER;Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).While a number of methods exist to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H., and Lipton, D., SIAM J. AppliedMath (1988) 48:1073). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in GUIDE TO HUGE COMPUTERS, Martin J. Bishop, ed., AcademicPress, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. AppliedMath (1988) 48:1073. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCS program package (Devereux, J., et al., Nucleic AcidsResearch (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. etal., J. Molec. Biol. (1990) 215:403).

As an illustration, by a polynucleotide having a nucleotide sequencehaving at least, for example, 95% “identity” to a reference nucleotidesequence encoding a polypeptide of a certain sequence it is intendedthat the nucleotide sequence of the polynucleotide is identical to thereference sequence except that the polynucleotide sequence may includeup to five point mutations per each 100 nucleotides of the referencepolypeptide sequence. Hence, the percentage of identity of a nucleotidesequence to a reference nucleotide sequence is to be calculated over thefull length of the reference nucleotide sequence. In other words, toobtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted and/or substitutedwith another nucleotide, and/or a number of nucleotides up to 5% of thetotal nucleotides in the reference sequence may be inserted into thereference sequence. These mutations of the reference sequence may occurat the 5′ or 3′ terminal positions of the reference nucleotide sequence,or anywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

Similarly, by a polypeptide having an amino acid sequence having atleast, for example, 95% “identity” to a reference amino acid sequence ofSEQ ID NO: 1 or 2 is intended that the amino acid sequence of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 1 or 2.Hence, the percentage of identity of an amino acid sequence to areference amino acid sequence is to be calculated over the full lengthof the reference amino acid sequence. In other words, to obtain apolypeptide having an amino acid sequence at least 95% identical to areference amino acid sequence, up to 5% of the amino acid residues inthe reference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at theamino- or carboxy-terminal positions of the reference amino acidsequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

A “nucleic acid” or “nucleic acid sequence” according to the presentinvention may include any polymer or oligomer of pyrimidine and purinebases, preferably cytosine, thymine, and uracil, and adenine andguanine, respectively (See Albert L. Lehninger, Principles ofBiochemistry, at 793-800 (Worth Pub. 1982), which is herein incorporatedby reference in its entirety for all purposes). The present inventioncontemplates any deoxyribonucleotide, ribonucleotide or peptide nucleicacid component, and any chemical variants thereof, such as methylated,hydroxymethylated or glycosylated forms of these bases, and the like.The polymers or oligomers may be heterogenous or homogenous incomposition, and may be isolated from naturally occurring sources or maybe artificially or synthetically produced. In addition, the nucleicacids may be DNA or RNA, or a mixture thereof, and may exist permanentlyor transitionally in single-stranded or double-stranded form, includinghomoduplex, heteroduplex, and hybrid states.

As used herein, the term “promoter” refers to a nucleic acid sequencethat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequences,including, but not limited to transcription factor binding sites,repressor and activator protein binding sites, and any other sequencesof nucleotides known to one of skill in the art to act directly orindirectly to regulate the amount of transcription from the promoter.Optionally the term “promoter” includes herein also the 5′ UTR region(5′ Untranslated Region) (e.g. the promoter may herein include one ormore parts upstream (5′) of the translation initiation codon of a gene,as this region may have a role in regulating transcription and/ortranslation). A “constitutive” promoter is a promoter that is active inmost tissues under most physiological and developmental conditions. An“inducible” promoter is a promoter that is physiologically (e.g. byexternal application of certain compounds) or developmentally regulated.A “tissue specific” promoter is only active in specific types of tissuesor cells. A “promoter active in a particular cell type, e.g. in T cells”refers to the general capability of the promoter to drive transcriptionwithin such a cell type. It does not make any implications about thespatio-temporal activity of the promoter.

The terms “amino acid sequence” or “protein” or “peptide” refer tomolecules consisting of a chain of amino acids, without reference to aspecific mode of action, size, 3 dimensional structure or origin. A“fragment” or “portion” of thereof may thus still be referred to as an“amino acid sequence” or “protein” or “peptide”. An “isolated amino acidsequence” is used to refer to an amino acid sequence which is no longerin its natural environment, for example in vitro or in a recombinantbacterial or human host cell.

“Engineered cells” refers herein to cells having been engineered, e.g.by the introduction of an exogenous nucleic acid sequence or specificalteration of an endogenous gene sequence. Such a cell has beengenetically modified for example by the introduction of for example oneor more mutations, insertions and/or deletions in the endogenous geneand/or insertion of a genetic construct in the genome. An engineeredcell may refer to a cell in isolation or in culture. Engineered cellsmay be “transduced cells” wherein the cells have been infected with e.g.a modified virus, for example, a retrovirus may be used, such asdescribed in the examples, but other suitable viruses may also becontemplated such as lentiviruses. Non-viral methods may also be used,such as transfections. Engineered cells may thus also be “stablytransfected cells” or “transiently transfected cells”. Transfectionrefers to non-viral methods to transfer DNA (or RNA) to cells such thata gene is expressed. Transfection methods are widely known in the art,such as calciumphosphate transfection, PEG transfection, and liposomalor lipoplex transfection of nucleic acids. Such a transfection may betransient, but may also be a stable transfection wherein cells can beselected that have the gene construct integrated in their genome.

“Randomly modifying” includes generating random sequences, and alsoincludes selecting δ2-CDR3 encoding regions and/or γ9-CDR-3 encodingregions that are occurring in nature and can be derived from γ9δ2T cellreceptor sequences derived from subjects, e.g. from humans, such asdescribed in the example section wherein such γ9-CDR3 and δ2-CDR3sequences were selected from a database. Generating random sequencesincludes modifying one or several amino acids in a starting γ9-CDR-3 andδ2-CDR3 sequence, up to the point wherein all the amino acids from thestarting sequences may be modified. Modifying amino acid sequences maybe done by changing nucleotide sequences of a codon such that the aminoacid encoded by that codon is altered.

The term “corresponding to” with regard to amino acid sequences meansthat when one sequence is aligned with a reference sequence whichcomprises a corresponding sequence, for example amino acid residues50-70, the amino acids aligning with the example amino acid residues50-70 are the amino acid residues of the one sequence that arecorresponding. An example of an alignment in which corresponding aminoacids are aligned from γ9-CDR3 and δ2-CDR3 regions is depicted in table3.

The term “selectable marker” is a term familiar to one of ordinary skillin the art and is used herein to describe any genetic entity which, whenexpressed, can be used to select for a cell or cells containing theselectable marker. Selectable marker gene products confer for exampleantibiotic resistance, or another selectable trait or a nutritionalrequirement. Selectable markers such as well-known in the art includegreen fluorescent protein (GFP), eGFP, luciferase, GUS and the like.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the invention relates to a method for identifyingγ9δ2T-cell receptors that mediate anti-tumour responses comprising thesteps of:

-   -   a) providing T-cells;    -   b) providing a nucleic acid sequence encoding a γ9-T-cell        receptor chain comprising a γ9-CDR3 encoding region, wherein the        γ9-CDR3 encoding region is randomly modified, and a nucleic acid        sequence encoding a δ2-T-cell receptor chain comprising a        δ2-CDR3 encoding region, wherein the δ2-CDR3 encoding region is        randomly modified;    -   c) introducing the nucleic acid sequences of step b) into the        T-cells to provide for an engineered T-cell with a γ9δ2T-cell        receptor comprising the γ9-T-cell receptor chain of step b) and        the δ2-T-cell receptor chain of step b);    -   d) optionally, repeating steps b) and c);    -   e) determining the anti-tumour response of the engineered        T-cells provided in steps c) and d);    -   f) identifying the γ9δ2T-cell receptors of the engineered        T-cells that mediate anti-tumour responses.

T cells, or T lymphocytes, belong to a group of white blood cells namedlymphocytes, which play a role in cell-mediated immunity. T cellsoriginate from hematopoietic stem cells in the bone marrow, mature inthe thymus (that is where the T is derived from), and gain their fullfunction in peripheral lymphoid tissues. During T-cell development,CD4⁻CD8⁻ T-cells (negative for both the CD4 and CD8 co-receptor) arecommitted either to an αβ or γδ fate as a result of an initial β or δTCR gene rearrangement. Cells that undergo early β chain rearrangementexpress a pre-TCR structure composed of a complete β chain and apre-TCRα chain on the cell surface. Such cells switch to a CD4⁺CD8⁺state, rearrange the TCRα chain locus, and express a mature αβTCR on thesurface. CD4⁻CD8⁻ T cells that successfully complete the γ generearrangement before the β gene rearrangement express a functional γδTCRand remain CD4⁻CD8⁻. (Claudio Tripodo et al. Gamm delta T cell lymphomasNature Reviews Clinical Oncology 6, 707-717 (December 2009). The T cellreceptor associates with the CD3 protein complex. Mature T cells, i.e.expressing a αβTCR or a γδTCR, express the T cell receptor complex onthe cell surface. The γδT-cells, which constitute about 1-5% of thetotal population of T cells, can be divided in further subpopulations. Asubpopulation of γδT-cells constitutes γ9δ2T-cells, which express aγ9δ2TCR. Within the extracellular domain of a T cell receptor threecomplementarity determining regions (CDR1, CDR2, CDR3) are located.These regions are in general the most variable domains and contributesignificantly to the diversity among TCRs. CDR regions are composedduring the development of a T-cell where so-called Variable-(V),Diverse-(D), and Joining-(J)-gene segments are randomly combined togenerate diverse TCRs.

αβT cells may be defined with respect of function as T lymphocytes thatexpress an αβTCR, which recognises peptides bound to MHC molecules(major histocompatibility complex), which are expressed on the surfaceof various cells. MHCs present peptides derived from the proteins of acell. When for example a cell is infected with a virus, the MHC willpresent viral peptides, and the interaction between the αβTCR and theMHC-complex activates specific types of T-cells which initiate andimmune responses to eliminate the infected cell. Hence, αβT cells may befunctionally defined as being cells capable of recognizing peptidesbound to MHC molecules. αβT cells may be selected from peripheral bloodfor example via the CD3 antigen as described below and in the examples,as the large majority of T cells have the αβTCR. αβT cells may also beselected with an antibody specific for the αβTCR, such as describedbelow. From such selected cells, the nucleic acid (or amino acid)sequence corresponding to the αT-cell receptor chain and the βT-cellreceptor chain may be determined. Hence, αβT-cells may also be definedas being cells comprising a nucleic acid (or amino acid) sequencecorresponding to the αT-cell receptor chain and/or the βT-cell receptorchain.

γ9δ2T-cells may be functionally defined in that they are specificallyand rapidly activated by a set of non-peptidic phosphorylated isoprenoidprecursors, collectively named phosphoantigens. Phosphoantigens areproduced by virtually all living cells. The most common phosphoantigenfound in animal and human cells (including cancer cells) is isopentenylpyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP).Activation of γ9δ2T-cells comprises clonal expansion, cytoxic activityand expression of cytokine. γ9δ2T-cells are also defined by expressionof the γ9δ2T-cell receptor. For example, cells may be selected using anantibody specific for the γ9δ2T-cell receptor such as described below.From such selected cells, the nucleic acid (or amino acid sequence)sequence corresponding to the γ9T-cell receptor chain and/or theδ2T-cell receptor chain may be determined. Hence, γ9δ2T-cells may alsobe defined as being cells comprising a nucleic acid (or amino acid)sequence corresponding to a γ9T-cell receptor chain and/or a δ2T-cellreceptor chain.

The person skilled in the art is well capable of selecting and/oridentifying cell populations characterized by expression of an antigenor receptor on the surface of the cell such as described throughoutherein. It is understood that with regard to expression on the surfaceof cells, such as CD3, CD4, CD8, αβTCR, γδTCR and γ9δ2TCR, this istypically done in a population of cells of which a portion of cells havea much higher level of expression of the antigen when compared to cellshaving a lower level of expression. Hence, the terms positive ornegative are to be understood as being relative, i.e. positive cellshave a much higher expression level as compared to cells being negative.Cells being negative in this sense may thus still have an expressionlevel which may be detected. Expression on the surface of cells may beanalysed using Fluorescence Activated Cell Sorting (FACS), and manyspecific antibodies are commercially available, e.g. such as for CD3,CD4, CD8, αβTCR, γδTCR and γ9δ2TCR, that are suitable for such FACSanalysis, such as described in the examples and as available.γ9δ2T-cells can hence also be defined and selected as being positive forγ9δ2TCR in FACS. Antibodies suitable for FACS or similar separationtechniques (such as e.g. antibodies conjugated to magnetic beads) arewidely available. Conditions are selected, such as provided by theantibody manufacturer that allows the selection of negative and/orpositive cells. Examples of antibodies that may be suitable forselection of γ9δ2-T cells, or engineered γ9δ2T cells such as availablefrom BD Pharmingen (BD, 1 Becton Drive, Franklin Lakes, N.J. USA) areVγ9-PE (clone B3, #555733), Vβ2-FITC (clone B6, #555738), γδTCRAPC(clone B1, #555718) or such as available from Beckman Coulter ispan-γδTCR-PE (clone IMMU510, # IM1418U) Similarly, suitable antibodiesfor αβ-T cell selection, such as anti-CD3 antibodies may be such asavailable from BD Pharmingen is CD3-FITC (#345763) or such as anti-αβTCRantibodies such as available from Beckman Coulter is pan-αβTCR-PE(#A39499) or pan-αβTCR-PC5 (#A39500).

Accordingly, in the method of the invention, first T-cells are provided.The T-cells may be primary cells, for example from a subject, such asdescribed in the examples for a human subject. The T-cells may be αβT-cells. The T-cells may also be cell lines, such as SupT-1, Jurkat, orRaji cells or any other widely available cell line. Any cell type, beinga primary cell or any other cell line will suffice, as long as the cellpopulation, or a substantial part thereof, expresses the T-cellreceptor, i.e. such as being positive for the αβT-cell receptor in aFACS sorting or the like as described above, such a cell population maybe contemplated. Also, any cell or cell population may be contemplatedthat, when provided with a γ9δ2TCR receptor according to the inventionis capable of forming a functional TCR complex and exerting e.g. afunctional cytoxic response and/or cytokine production. The cell that isprovided may also be a progenitor cell, preferably a blood progenitorcell such as a thymocyte or a blood stem cell, which after it has beenprovided with the right stimuli can develop into T cells or engineered Tcells. Hence it is understood that providing T cells and providing thesewith the γ9δ2TCR receptor according to the invention may also compriseproviding progenitor cells, providing these with the γ9δ2TCR receptorand stimulating these progenitor cells such that these develop intoengineered T-cells.

Also, a nucleic acid sequence is provided encoding a γ9-T-cell receptorchain wherein the γ9-CDR3 encoding region is randomly modified and anucleic acid sequence encoding a δ2-T-cell receptor chain wherein theδ2-CDR3 encoding region is randomly modified. Preferably, the γ9-T-cellreceptor chain and the δ2-T-cell receptor chain are of human origin. Thehuman CDR3 regions of γ9 and δ2 are well defined. For example, the humanCDR3 region of the γ9-TCR chain of amino acid sequence SEQ ID NO.1corresponds to amino acid residues 118-132, which corresponds to aminoacid residues γ105-γ117 according to the International ImmunogeneticsInformation System (IMGT). The human CDR3 region of the δ2 TCR chain ofamino acid sequence SEQ ID NO.2 corresponds to amino acid residues112-127 which corresponds to amino acid residues δ105-δ117 according tothe IMGT. According to the Internation Immunogenetics Information System(IMGT) (Lefranc MP. IMGT, the international ImMunoGeneTics database.Nucleic Acids Res. 2003; 31:307-310, which is incorporated herein byreference) the human CDR3 region may be delimited by, but does notinclude, the anchor positions C104 and F118, see also table 3, which arereferred to for each chain as γC104 and γF118 and δC104 and δF118. Theanchor positions γC104 corresponds to C117 in SEQ ID NO.1 and γF118corresponds to F133 in SEQ ID NO.1. The anchor position δC104corresponds to C111 of amino acid sequence SEQ ID NO.2 and the anchorposition δF118 corresponds to F128 of amino acid sequence SEQ ID NO.2.The IMGT provides a common access to sequence, genome and structure forinstance of T cell receptors of different species including human T cellreceptors and allows the identification of a γ9T-cell receptor chain anda δ2T-cell receptor chain including the CDR3 regions, e.g. via theanchor positions.

According to the IMGT, the CDR3 is delimited by (but does not include)the anchor positions 2nd-CYS 104 and J-PHE or J-TRP 118. The JUNCTIONincludes 2nd-CYS 104 and J-PHE or J-TRP 118 and is therefore two aminoacids longer than the CDR3. The CDR3 numbering goes from 105 to 117 and,if necessary, gaps or additional positions are added at the top of theloop. Note that, the J-PHE or J-TRP belongs to the characteristicJ-REGION motif ‘F/W-G-X-G’ at positions 118-121, and that the CDR3 isdelimited by the same anchor positions (2nd-CYS 104 and J-PHE or J-TRP118), whatever the receptor type (IG or TR), the chain type (heavy orlight for IG; alpha, beta, gamma or delta for TR) or the species. Seealsohttp://www.imgt.org/IMGTScientificChart/Numbering/IMGTIGVLsuperfamily.htmlfor an explanation of the IMGT numbering. This IMGT numbering is alsoused in the examples.

Hence, by using the anchor positions, according to the IMGT standards,CDR3 regions can easily be identified. For example via entering an aminoacid sequence (or nucleic acid sequence) using available online toolssuch as provided by IMGT (for example IMGT/V-QUEST) a γ9T-cell receptorchain sequence or a δ2T-cell receptor chain sequence can easily beidentified as well as the exact CDR3 regions. Alternatively, γ9T-cellreceptor chain sequences or δ2T-cell receptor chain sequences alreadypublicly available can be found in the IMGT and hence, the CDR3 regionscan be easily identified therefrom. Alternatively, using SEQ ID NO.1 andSEQ ID No.2 as a reference sequence and the anchor amino acid residuepositions therein, the CDR3 regions of other γ9-T-cell and δ2-T-cellreceptor chains are easily identified via an alignment, such as depictedfor different CDR3 region in table 3. Hence, the skilled person caneasily identify any CDR3 region from any γ9T-cell receptor chainsequence that corresponds with the CDR3 region from SEQ ID NO.1, andalso the skilled person can easily identify any CDR3 region from anyδ2T-cell receptor chain sequence that corresponds with the CDR3 regionsfrom SEQ ID NO.2. Hence, the skilled person knows which amino acidsequence from any γ9-T-cell and/or δ2-T-cell receptor chains he needs torandomly modify.

Hence, modifying the δ2-CDR3 encoding region in a δ2-T-cell receptorchain nucleic acid sequence may comprise aligning the nucleic acidsequence of SEQ ID NO.4 to the δ2-T-cell receptor chain nucleic acidsequence of interest, identifying the codons in the δ2-T-cell receptorchain nucleic acid sequence corresponding to the codons encoding theanchor positions in SEQ ID NO.4, and randomly modifying the nucleic acidsequence in between the anchor positions codons identified in theδ2-T-cell receptor chain nucleic acid sequence of interest. Also,modifying the γ9-CDR3 encoding region in a γ9-T-cell receptor chainnucleic acid sequence may comprises aligning the nucleic acid sequenceof SEQ ID NO.4 to the γ9-T-cell receptor chain nucleic acid sequence ofinterest, identifying the codons in the γ9-T-cell receptor chain nucleicacid sequence corresponding to the codons encoding the anchor positionsin SEQ ID NO.3, and randomly modifying the nucleic acid sequence inbetween the anchor positions codons identified in the γ9-T-cell receptorchain nucleic acid sequence of interest. The random modification of thenucleic acid sequence of the CDR3 encoding regions is such that theamino acid sequence encoded by the nucleic acid sequence is modified,i.e. at least one amino acid residue is changed in another amino acidresidue, or at least one amino acid residue is inserted or deleted.

The nucleic acid sequences encoding the γ9-T-cell receptor chain and theδ2-T-cell receptor chain may be introduced into T-cells to provide foran engineered T-cell with a γ9δ2T-cell receptor comprising the γ9-T-cellreceptor chain and the δ2-T-cell receptor chain. By expressing theγ9-T-cell receptor chain and the δ2-T-cell receptor chain of which theCDR3 regions have been randomly modified, a specific γ9δ2-T-cellreceptor is expressed in the cell. Optionally, the steps of providing aCDR3 randomly modified γ9-T-cell receptor chain and δ2-T-cell receptorchain and subsequent introduction into T-cells may be repeated. Forexample, each time this is done a different combination of randomlymodified CD3 regions is used. It is also envisioned that the steps ofrepeating the steps, e.g. with different combinations of randomlymodified CD3 regions, may be performed, at least in part,simultaneously. For example, a plurality of nucleic acid sequencesencoding a γ9-T-cell receptor chain wherein the γ9-CDR3 encoding regionis randomly modified and a nucleic acid sequence encoding a δ2-T-cellreceptor chain wherein the δ2-CDR3 encoding region is randomly modified,may be provided, and this plurality of nucleic acid sequences may beintroduced in T-cells thereby providing a plurality of engineeredT-cells.

The anti-tumour response of the provided engineered T-cell expressing aγ9δ2T-cell receptor or an engineered γ9δ2T-cell receptor that mediateanti-tumour responses is identified. In case the steps of providing thenucleic acid sequences and introduction into the T-cells is performed inseparate steps, the step of determining the anti-tumour responses andidentifying the engineered γ9δ2T-cell receptor may be combined becauseit is known which nucleic acid sequences were introduced in each of thecorresponding engineered γ9δ2T-cell. Determining the anti-tumourresponses may hence be performed for each engineered γ9δ2T-cellseparately. Alternatively, for example when engineered γ9δ2T-cells arecombined, e.g. when repeating the steps such as described above isperformed simultaneously, and a plurality of engineered T-cells isprovided, the engineered T-cells may be separated and the anti-tumourresponse determined for each of the separated engineered T-cells. Theengineered T-cells may be separated over different compartments and ineach of the compartments the anti-tumour response may be determined. Insuch a scenario, the step of identifying the engineered T-cells thatmediate anti-tumour responses may involve determining the sequence, e.g.via sequencing, of the nucleic acid sequence encoding each of the CDR3regions of the γ9δ2TCR. Hence, optionally as a final step, identifyingengineered T-cells that mediate anti-tumour responses involvesdetermining the nucleic acid sequence encoding the γ9-T-cell receptorchain comprising the γ9-CDR3 encoding region and the nucleic acidsequence encoding the δ2-T-cell receptor chain comprising the δ2-CDR3encoding region. It may also be envisioned to only determine the nucleicacid sequence of both of the γ9-CDR3 and the δ2-CDR3 encoding regions,i.e. it is not required to determine the complete nucleic acid sequencesof the γ9-T-cell receptor chain and/or the δ2-T-cell receptor chain. Asan alternative to determining the nucleic acid sequence, the amino acidsequences of the γ9-CDR3 and the δ2-CDR3 region may also be determined.

Preferably, the nucleic acid sequence encoding a γ9-T-cell receptorchain encodes an amino acid sequence having at least 60% sequenceidentity with the amino acid sequence of SEQ ID NO.1, and wherein thenucleic acid sequence encoding a δ2-T-cell receptor chain encodes anamino acid sequence having at least 60% sequence identity with the aminoacid sequence of SEQ ID NO.2. The nucleic acid sequence encoding aγ9-T-cell receptor chain comprises preferably a nucleic acid sequenceencoding an amino acid sequence having at least 70, 80, 90, 95 or 99%sequence identity with the amino acid sequence of SEQ ID NO.1. Thenucleic acid sequence encoding a δ2-T-cell receptor chain comprisespreferably a nucleic acid sequence encoding an amino acid sequencehaving at least 70, 80, 90, 95 or 99% sequence identity with the aminoacid sequence of SEQ ID NO.2. The skilled person is capable of defininga γ9δ2-T-cell receptor with respect to function. Preferably, thepercentage of sequence identity is calculated over the entire length ofSEQ ID NO.1 or SEQ ID NO.2.

Random modification of the CDR3 regions of the γ9-CDR3 and δ2-CDR3 maycomprise the arbitrary selection of amino acid residues. Such arbitraryselection may be done by generating random sequences in vitro, e.g. byrandom chemical synthesis, such as provided by Sloning Biotechnologyusing chemical synthesis (Sloning BioTechnology GmbH-A Division ofMorphoSys, Zeppelinstrasse 4, 82178 Puchheim, Germany). Randommodification of the CDR3 regions of the γ9-CDR3 and δ2-CDR3 may alsocomprise the arbitrary selection from sequences found in natural CDR3regions. Such natural CDR3 amino acid sequences are generated randomly,i.e. arbitrarily selected, by nature.

With regard to the random modification, the randomly modified γ9-CDR3encoding region may preferably be modified at the amino acid sequencecorresponding to amino acid residues 118-132 of SEQ ID NO 1. With regardto the random modification, the randomly modified γ9-CDR3 encodingregion may preferably be modified at the amino acid sequencecorresponding to amino acid residues 122-124 of SEQ ID NO 1. As said, aγ9-CDR3 encoding region is easily identified, e.g. via alignment and/orfrom information as provided by IMGT, and hence the amino acid residuesfrom a γ9-CDR3 encoding region corresponding to the amino acid residues118-132 of SEQ ID NO.1 as well. Optionally, amino acid residues C117 andF133 of SEQ ID NO.1 may further be used for identifying thecorresponding amino acid residues. In an alignment, such as depicted intable 3, the amino acid residues of a γ9-CDR3 encoding regioncorresponding to amino acid residues 118-132 of amino acid sequence SEQID NO.1 can easily be identified, and thus amino acid residuescorresponding to amino acid residues 122-124 of SEQ ID NO 1 as well.Hence, a corresponding sequence may be easily be identified via aligningthe amino acid sequence of interest to the reference amino acid sequence(SEQ ID NO.1), and identifying the anchor amino acid residues C117 andF133, so that the CDR3 region is identified. Next, the CDR3 regions arealigned and the amino acid residues of the amino acid sequence thatcorrespond to 118-132 of SEQ ID NO.1 is identified. With regard to therandom modification, the randomly modified δ2-CDR3 encoding region maybe modified at the amino acid sequence corresponding to amino acidresidues 115-122 of SEQ ID NO.2. With regard to the random modification,the randomly modified δ2-CDR3 encoding region may be modified at theamino acid sequence corresponding to amino acid residues 112-127 of SEQID NO.2. A δ2-CDR3 encoding region may be easily identified, e.g. viaalignment and/or from information as provided by IMGT, and hence theamino acid residues from a δ2-CDR3 encoding region corresponding to theamino acid residues 112-127 of SEQ ID NO.2 as well. Optionally, aminoacid residues C111 and F128 of SEQ ID NO.2 may further be used foridentifying the corresponding amino acid residues. In an alignment, suchas depicted in table 3, the amino acid residues of a δ2CDR3 encodingregion corresponding to amino acid residues 112-127 of amino acidsequence SEQ ID NO.2 can easily be identified. Hence, a correspondingsequence may be easily identified via aligning the amino acid sequenceof interest to the reference amino acid sequence (SEQ ID NO.2), andidentifying the anchor amino acid residues C111 and F128, such that theCDR3 region is identified. Next, the CDR3 regions are aligned and theamino acid residues of the amino acid sequence that correspond to112-127 of SEQ ID NO.2 can bes identified, and thus the amino acidsequence that correspond to amino acid residues 115-122 as well.

In one embodiment, randomly modifying the amino acid sequencecorresponding to amino acid residues 115-122 of SEQ ID NO.2 comprises amodification in length.

In one embodiment, randomly modifying the amino acid sequencecorresponding to amino acid residues 112-127 of SEQ ID NO.2 comprises amodification in length.

In one embodiment, randomly modifying the amino acid sequence comprisesintroducing amino acid substitutions, deletions, and/or insertions.

With regard to the random modification, this may include anymodification such as amino acid substitution, deletion and/or insertion.Hence, the random modification may include a modification in sequenceand/or length. The CDR3 regions may thus vary in length ranging from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21or 22 amino acid residues. Preferably, the δ2-CDR3 encoding region mayvary in length ranging from 5-7 amino acids between positions δL109 andδT113 according to the IGMT annotation. Hence, preferably the δ2-CDR3encoding region corresponding to amino acid sequence 112-127 of SEQ IDNO.2 is randomly modified between L116 and T123 of SEQ ID NO.2 such thatit contains 5-7 amino acids in this region. Hence, the entire δ2-CDR3encoding region preferably varies in length ranging from 15-17 aminoacid residues. Amino acid substitution may include any substitution suchas conserved substitutions wherein one amino acid from a group ischanged for another amino acid residue of the group, e.g. one aliphaticamino acid residue is exchange for another aliphatic amino acid residue,or a non-conserved substitution wherein one amino acid from a group ischanged for another amino acid residue of a different group, e.g. onealiphatic amino acid residue is exchange for an basic amino acidresidue.

With regard to providing a nucleic acid, it is understood that thenucleic acid is provided such that when it is introduced in a cell thatit can be expressed such that the amino acid sequence it encodes isexpressed on the surface of the cell. Preferably this is done byintegrating the nucleic acid or nucleic acids into the genome of thecell. Preferably, the nucleic acid sequence encoding a γ9-T-cellreceptor chain is provided in a genetic construct and the nucleic acidsequence encoding a δ2-T-cell receptor chain is provided in a geneticconstruct. The genetic constructs may be provided on separate vectors.The genetic constructs may also be provided on a single vector. Thenucleic acid sequence encoding a γ9-T-cell receptor chain and thenucleic acid sequence encoding a δ2-T-cell receptor chain may also beprovided in a single genetic construct. For example, a single geneticconstruct may express a single mRNA comprising an Internal RibosomalEntry Site, such as described in the example, such that each receptorchain may be transcribed from the same mRNA.

The genetic construct and/or vectors may also comprise selectablemarkers. A selectable marker may be defined as any nucleic acid sequenceand/or amino acid sequence that allows cells that are provided therewithto be selected. For example, selectable markers may be neomycine orpuromycin resistance genes such as described in the examples. Selectionof cells to which the genetic construct and/or vector has beentransferred may than be performed by incubating in the presence ofneomycine or puromycin. Other selectable markers may be for example anyone of green, red and yellow fluorescent proteins. Selecting may than beperformed by using FACS. It is not required to have a selectable marker,as the cell when expressing the γ9δ2T-cell receptor may be selectedbased on that expression on itself, e.g. via selection with an antibodydirected thereto such as described above. It may be provided that thehost cell does not express a substantial amount of the γ9δ2T-cellreceptor to allow selection of engineered γ9δ2T-cells, i.e. expressionof the endogenous γ9δ2-T-cell receptor must be much lower as comparedwith the engineered γ9δ2-T-cell receptor.

Preferably, the nucleic acid sequence encoding a γ9-T-cell receptorchain is provided in a retroviral vector and the nucleic acid sequenceencoding a δ2-T-cell receptor chain is provided in a retroviral vectorand the steps of introducing the nucleic acid sequences into the T-cellscomprises retroviral vector transduction of the T-cells. Thetransduction may be performed simultaneously or in subsequent steps.Alternatively, the nucleic acid sequence encoding a γ9-T-cell receptorchain and the nucleic acid sequence encoding a δ2-T-cell receptor chainare provided in a single retroviral vector, and the steps of introducingthe nucleic acid sequences into the T-cells comprises retroviral vectortransduction of the T-cells. Retroviral vectors, such as described inthe examples, are highly efficient for transferring the nucleic acidsequences to the T-cells such that engineered T-cells can be provided.Many retroviral and lentiviral vectors are known or such as described inthe examples. Retroviral vectors have an RNA genome which, when enteredin a cell, is reverse transcribed into DNA that is subsequentlyintegrated into the host genome. Integration is advantageous as itallows to proliferation of transduced cells while maintaining the viralvector genome comprising the genetic construct.

In one embodiment, the step of determining the anti-tumour reactivitycomprises contacting the cells with tumour cells. Tumour cells may beany kind of tumour cells. For example, primary tumour cells from apatient. The tumour cells may be tumour cells from cell lines, such asthe cell lines listed in the examples named Daudi, RPMI8226/S, OPM2,LME1, K562, Saos2, MZ1851RC, SCC9, Fadu, MDA-MB231, MCF7, BT549, SW480,which are well known in the art. Tumour cell lines may easily beobtained from the American Type Culture Collection (ATCC, Manassas, Va.)and the like. The step of determining anti-tumour activity may includeany assay in which an anti-tumour effect may be determined, such ashaving an effect on tumour cell division rate, i.e. the speed with whichthe tumour cells divide, cell death, binding to the tumour cells etc.

Determining the anti-tumour responses includes contacting the engineeredT-cell with a tumour cell and measuring its ability to lyse the tumourcell and/or induce IFN-γ production. The ability to lyse the tumourcells include providing a fixed amount of tumour cells with which theengineered γ9δ2 T-cell, i.e. an engineered T-cell expressing a γ9δ2TCR,is contacted and after an incubation period the number of viable tumourcells is counted. When the number of viable cells counted is compared toa control not contacted with the engineered γ9δ2 T-cell, and the numberis lower, such an engineered γ9δ2 T-cell can be regarded to have ananti-tumour response. In addition to counting the viable cells, one mayalso perform a ⁵¹Chromium-release assay similarly to what is describedin the examples. The amount of ⁵¹Chromium release being a measure of thenumber of cells that have been lysed.

Similarly, IFN-γ production may also be determined, e.g. via antibodystaining, ELISA and/or quantitative PCR for the expressed mRNA. Assaysfor determining IFN-γ are commercially widely available, such asdescribed in the example. Engineered γ9δ2 T-cells are contacted with thetumour cells. The contacting may be in the presence of a phosphoantigen,such as pamidronate. Hence, when the amount of IFN-γ produced is higheras compared to when cells are not contacted with the engineered γ9δ2T-cell, such an engineered γ9δ2 T-cell may be regarded as having ananti-tumour response. In addition to comparing with a control, i.e. tocells not contacted with the engineered γ9δ2 T-cell, one may alsocompare with a reference value. In any case, any assay in which aneffect on tumour cells may be determined involving contacting the γ9δ2T-cell with the tumour cell may be contemplated. As long as ananti-tumour effect, such as induction of cell death, cell viability,binding to the tumour cell, and/or IFN-γ production may be determined.

Furthermore, with regard to the methods described above, the T cells maybe expanded before or after the transfer of the nucleic acids encodingthe engineered γ9δ2 T-cell receptor, i.e. with the randomly modifiedCDR3 regions. Preferably, the expansion is after the transfer such thatrelatively little nucleic acids need to be transferred. This expansionof the T cells may be performed by stimulation with α-CD3/CD28 Dynabeadsin the presence of IL-2. A rapid expansion protocol such as described inthe examples may also be used. The expanded cells comprising theengineered γ9δ2 T-cell receptor, which may be selected e.g. via aselectable marker such as described above, may be further selected forthe presence of the CD4 antigen and the CD8 antigen, e.g. using the MACSseparating system as described in the examples. The engineered T-cellsmay be subsequently further expanded using the REP protocol as describedby Riddel and Greenberg, 1990 J Immunol Methods. 128(2):189-201, whichis incorporated herein by reference, or using similar further expansionmethods thereto. Briefly, the expansion method involves using antibodiesdirected against T cell activation molecules, such as TCR, CD3 and CD28and/or feeder cells and/or stimulating cytokines.

In another embodiment, a γ9δ2T-cell receptor, or a fragment thereof, isprovided comprising a γ9-T-cell receptor chain comprising a γ9-CDR3region, wherein the amino acid sequence of the γ9-CDR3 regioncorresponding to amino acid residues 122-124 of SEQ ID NO.1 is modifiedand a δ2-T-cell receptor chain comprising a δ2-CDR3 region wherein theamino acid sequence of the δ2-CDR3 region corresponding to amino acidresidues 115-122 of SEQ ID NO.2 is modified, wherein the combinations ofthe amino acid modifications within the γ9δ2T-cell receptor, or afragment thereof, are as listed in Table 1. The substitutions maycomprise the combinations such as listed in table 1. The amino acidsequences of the respective γ9-CDR3 and δ2-CDR3 regions are substitutedwith amino acid sequences selected from the group consisting ofcombinations of amino acid sequences AQQ (γ9) and ALKRTD (δ2); AQQ (γ9)and LLGY(δ2); IQ (γ9) and ALKRTD (δ2); and; IQ (γ9) and TLGMGGEY(δ2).These γ9δ2T-cell receptors, or fragments thereof, comprising theseparticular combinations of CDR3 regions, were identified using themethods according to the invention such as described above and asdescribed in the examples.

Combinations of amino acid sequences of γ9-CDR3 and δ2-CDR3 regions thatmay be preferably combined are listed below in table 1 as they show ananti-tumour effect. Table 1. Combinations of amino acid sequences ofγ9-CDR3 and δ2-CDR3 regions. The region corresponding to amino acidresidues of the γ9-CDR3 region corresponding to number 122-124 of SEQ IDNO.1 which has been substituted, i.e. has been modified, is listed, andthe region corresponding to amino acid residues of the δ2-CDR3 regioncorresponding to amino acid residues 115-122 of SEQ ID NO.2 which hasbeen substituted is listed as well. The different combinations ofγ9-CDR3 and δ2-CDR3 are numbered from 1-4. For example, number 2corresponds to a γ9δ2TCR with a γ9-T-cell receptor chain wherein theγ9-CDR3 region corresponding to number 122-124 of SEQ ID NO.1 has beensubstituted with AQQ and a δ2T-cell receptor chain wherein the δ2-CDR3region corresponding to amino acid residues 115-122 of SEQ ID NO.2 hasbeen substituted with ALKRTD.

TABLE 1  Combinations of modified amino acid sequences ofγ9-CDR3 and δ2-CDR3 regions, wherein the listedamino acid sequences substitute γ9-CDR3 amino acidresidues corresponding with 122-124 of SEQ IDNO. 1 and substitute δ2-CDR3 amino acid residuescorresponding with 115-122 of SEQ ID NO. 2. Nr. combination γ9-CDR3δ2-CDR3 1 γ9-cl5/δ2-cl5 IQ ALKRTD (SEQ ID NO. 15) 2 γ9-G115/δ2-cl5 AQQALKRTD (SEQ ID NO. 15) 3 γ9-cl5/δ2-G115 IQ TLGMGGEY (SEQ ID NO. 19) 4γ9-G115/δ2-cl3 AQQ LLGY

In another embodiment, a γ9δ2T-cell receptor, or a fragment thereof, isprovided comprising a γ9-T-cell receptor chain comprising a γ9-CDR3region, wherein the amino acid sequence of the γ9-CDR3 regioncorresponding to amino acid residues 118-132 of SEQ ID NO.1 is modifiedand a δ2-T-cell receptor chain comprising a δ2-CDR3 region wherein theamino acid sequence of the δ2-CDR3 region corresponding to amino acidresidues 112-127 of SEQ ID NO.2 is modified, wherein the combinations ofthe amino acid modifications within the γ9δ2T-cell receptor, or afragment thereof, are as listed in Table 2. The substitutions maycomprise the combinations such as listed in table 2. The amino acidsequences of the respective γ9-CDR3 and δ2-CDR3 regions are substitutedwith amino acid sequences selected from the group consisting ofcombinations of amino acid sequences ALWEIQELGKKIKV (γ9) andACDALKRTDTDKLI (δ2); ALWEAQQELGKKIKV (γ9) and ACDALKRTDTDKLI (δ2);ALWEIQELGKKIKV (γ9) and ACDTLGMGGEYTDKLI (δ2); ALWEAQQELGKKIKV (γ9) andACDLLGYTDKLI (δ2). These γ9δ2T-cell receptors, or fragments thereof,comprising these particular combinations of CDR3 regions, wereidentified using the methods according to the invention such asdescribed above and as described in the examples.

Combinations of amino acid sequences of γ9-CDR3 and δ2-CDR3 regions thatmay be preferably combined are listed below in table 2 as they show astrong anti-tumour effect. Table 2. Combinations of amino acid sequencesof γ9-CDR3 and δ2-CDR3 regions. The region corresponding to amino acidresidues of the γ9-CDR3 region corresponding to number 118-132 of SEQ IDNO.1 which has been substituted, i.e. has been modified, is listed, andthe region corresponding to amino acid residues of the δ2-CDR3 regioncorresponding to amino acid residues 112-127 of SEQ ID NO.2 which hasbeen substituted, i.e. is modified, is listed as well. The differentcombinations of γ9-CDR3 and δ2-CDR3 are numbered from 1-4 in table 2.For example, number 2 corresponds to a γ9δ2TCR with a γ9-T-cell receptorchain wherein the γ9-CDR3 region corresponding to number 118-132 of SEQID NO.1 has been modified to (substituted with) ALWEAQQELGKKIKV and aδ2T-cell receptor chain wherein the δ2-CDR3 region corresponding toamino acid residues 112-127 of SEQ ID NO.2 has been modified to(substituted with) ACDALKRTDTDKLI.

TABLE 2  Combinations of complete modified amino acidsequences of γ9-CDR3 and δ2-CDR3 regions Nr. combination γ9-CDR3 δ2-CDR31 γ9-cl5/ ALWEIQELGKKIKV ACDALKRTDTDKLI δ2-cl5 (SEQ. ID NO. 16)(SEQ. ID NO. 17) 2 γ9-G115/ ALWEAQQELGKKIKV ACDALKRTDTDKLI δ2-cl5(SEQ. ID NO. 3) (SEQ. ID NO. 17) 3 γ9-cl5/ ALWEIQELGKKIKVACDTLGMGGEYTDKLI δ2-G115 (SEQ. ID NO. 16) (SEQ. ID NO. 4) 4 γ9-G115/ALWEAQQELGKKIKV ACDLLGYTDKLI δ2-cl3 (SEQ. ID NO. 3) (SEQ. ID NO. 18)

In one embodiment, the γ9δ2T-cell receptor, or a fragment thereof,comprises a γ9-T-cell receptor chain comprising a γ9-CDR3 region,wherein the amino acid sequence of the γ9-CDR3 region corresponding toamino acid residues 122-124 of the amino acid sequence of SEQ ID NO.1 isAQQ, and a δ2-T-cell receptor chain comprising a δ2-CDR3 region whereinthe amino acid sequence of the δ2-CDR3 region corresponding to aminoacid residues 115-122 of the amino acid sequence of SEQ ID NO.2 isALKRTD. In a further embodiment, the γ9δ2T-cell receptor, or a fragmentthereof, comprises a γ9-T-cell receptor chain comprising a γ9-CDR3region, wherein the amino acid sequence of the γ9-CDR3 regioncorresponding to amino acid residues 118-132 of the amino acid sequenceof SEQ ID NO.1 is ALWEAQQELGKKIKV, and a δ2-T-cell receptor chaincomprising a δ2-CDR3 region wherein the amino acid sequence of theδ2-CDR3 region corresponding to amino acid residues 112-128 of the aminoacid sequence of SEQ ID NO.2 is ACDALKRTDTDKLI. This particularcombination of CDR3 regions in the γ9δ2T-cell receptor, or fragmentthereof has shown a particular high anti-tumour activity and may hencebe preferred.

In one embodiment, the γ9δ2T-cell receptor or fragment thereof accordingto invention, wherein the amino acid sequence of the γ9-CDR3 regioncorresponding to amino acid residues 118-121 of the amino acid sequenceof SEQ ID NO.1 is ALWE, and wherein the amino acid sequence of theγ9-CDR3 region corresponding to amino acid residues 125-132 of the aminoacid sequence of SEQ ID NO.1 is ELGKKIKV, and wherein the amino acidsequence of the δ2-CDR3 region corresponding to amino acid residues112-114 of the amino acid sequence of SEQ ID NO.2. is ACD, and whereinthe amino acid sequence of the δ2-CDR3 region corresponding to aminoacid residues 123-127 of the amino acid sequence of SEQ ID NO.2 isTDKLI. It is understood that these amino acid sequences correspond tothe sequences which are flanking the amino acid sequences that are to besubstituted with the amino acid sequences as listed above.

In a further embodiment, a γ9δ2T-cell receptor, or fragment thereof, isprovided, wherein the γ9-T-cell receptor chain comprises an amino acidsequence having at least 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequenceidentity with the amino acid sequence of SEQ ID NO.1, and/or wherein theδ2-T-cell receptor chain comprises an amino acid sequence having atleast 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with theamino acid sequence of SEQ ID NO.2. It is understood that the amino acidsequence of the γ9-CDR3 region corresponding to amino acid residuesnumber 122-124 of SEQ ID NO.1, and the amino acid sequence of theδ2-CDR3 region corresponding to amino acid residues 115-122 of SEQ IDNO.2, are in this embodiment substituted in accordance with Table 1.

In one embodiment, a γ9δ2T-cell receptor, or fragment thereof isprovided, wherein the γ9-T-cell receptor chain comprises an amino acidsequence comprising amino acid residues 1-121 and 125-315 of the aminoacid sequence of SEQ ID NO.1 and wherein the modified amino acidresidues 122-124 are as defined Table 1, and wherein the δ2-T-cellreceptor chain comprises an amino acid sequence comprising amino acidresidues 1-114 and 123-292 of the amino acid sequence of SEQ ID NO.2 andwherein the modified amino acid residues 115-122 are as defined inTable 1. It is understood that the amino acid sequence of the γ9-CDR3region corresponding to amino acid residues number 122-124 of SEQ IDNO.1, and the amino acid sequence of the δ2-CDR3 region corresponding toamino acid residues 115-122 of SEQ ID NO.2, are in this embodimentsubstituted in accordance with Table 1.

It is understood that the amino acid sequences SEQ ID NO.1 and SEQ IDNO.2 each comprise a leader sequence. The leader sequence of amino acidsequence SEQ ID NO.1 is from amino acid Nr. 1-20 of SEQ ID NO.1. Theleader sequence of amino acid sequence SEQ ID NO.2 is from amino acidNr. 1-19 of SEQ ID NO.2. The leader sequence may directs the amino acidchain to the surface of the cell. The leader sequence can be cleavedfrom the nascent amino acid chain and is not present in the finalprotein. Hence, in one embodiment, a γ9δ2T-cell receptor, or fragmentthereof is provided, wherein the γ9-T-cell receptor chain comprises anamino acid sequence comprising amino acid residues 21-121 and 125-315 ofthe amino acid sequence of SEQ ID NO.1 and wherein the modified aminoacid residues 122-124 are as defined in Table 1, and wherein theδ2-T-cell receptor chain comprises an amino acid sequence comprisingamino acid residues 20-114 and 123-292 of the amino acid sequence of SEQID NO.2 and wherein the modified amino acid residues 115-122 are asdefined in Table 1. It is understood that the amino acid sequence of theγ9-CDR3 region corresponding to amino acid residues number 122-124 ofSEQ ID NO.1, and the amino acid sequence of the δ2-CDR3 regioncorresponding to amino acid residues 115-122 of SEQ ID NO.2, are in thisembodiment substituted in accordance with Table 1. It is also understoodthat it may be optional to use alternative leader sequences, and it isalso understood that this leader sequences from SEQ ID NO.1 and SEQ IDNO.2 may be disregarded, e.g. when comparing sequences and/determiningcorresponding sequences and/or alignment and/or determining percentagesof identity.

In another embodiment, a conjugate is provided comprising a solublefragment of the γ9δ2T-cell receptor according to any one of theγ9δ2T-cell receptors as described above. The extracellular domain of theγ9δ2-T-cell receptor comprises the amino acid sequence of the γ9-T-cellreceptor chain corresponding to amino acid residues 21-263 of the aminoacid sequence of SEQ ID NO.1 and the amino acid sequence of theδ2-T-cell receptor chain corresponding to amino acid residues 20-249 ofthe amino acid sequence of SEQ ID NO.2. The conjugate may be linked toan agent. Preferably, the agent is selected from the group consisting ofa diagnostic agent, a therapeutic agent, an anti-cancer agent, achemical, a nanoparticle, a chemotherapeutic agent or a fluorochrome.Such conjugates that may be linked to substrates (e.g. chemicals,nanoparticles) may be used e.g. to deliver chemotherapy to a target ofinterest. In addition, in diagnostics expression of defined ligands maybe tested by taking advantage of the soluble TCRs linked tofluorochromes which are then used as staining tool or for thebiochemical isolation of the ligand.

Furthermore, in this embodiment, the invention provides for isolatednucleic acid sequences encoding the δ2-T-cell receptor chain, orfragment thereof, according to the invention, wherein the δ2-CDR3 regioncorresponding to amino acid residues 115-122 of the amino acid sequenceSEQ ID NO.2 is substituted with amino acid residues ALKRTD or LLGY.Hence, the δ2-CDR3 region corresponding to amino acid residues 112-127of the amino acid sequence SEQ ID NO.2 is substituted between D114 andT123 of SEQ ID NO.2 with amino acid residues ALKRTD or LLGY. Theinvention further provides for an isolated nucleic acid sequenceencoding the γ9-T-cell receptor chain, or fragment thereof, according tothe invention, wherein the amino acid sequence of the γ9-CDR3 regioncorresponding to amino acid residues 122-124 of the amino acid sequenceSEQ ID NO.1 is substituted with amino acid residues IQ. Hence, theγ9-CDR3 region corresponding to amino acid residues 118-132 of the aminoacid sequence SEQ ID NO.1 is substituted between E121 and E 125 of SEQID NO 1 with amino acid residues IQ.

Also are provide genetic constructs comprising the isolated nucleic acidsequences according to the invention, and retroviral vectors comprisingthe genetic construct.

For example, isolated nucleic acid sequences are listed as SEQ ID NO.3and SEQ ID NO.4 and correspond respectively to open reading frames ofthe γ9-T-cell receptor chain (encoding corresponding amino acid sequenceSEQ ID NO.1) and the δ2-T-cell receptor chain (encoding correspondingamino acid sequence SEQ ID NO.2) of the G115wt clone, such as describedin the examples. These isolated nucleic acid sequences, when being partof an expression cassette may be interspersed by for example intronicencoding sequences, and have 5′ and 3′ non-coding regions. When suchsequences are expressed, the corresponding γ9-T-cell receptor chain andthe δ2-T-cell receptor chain amino acid sequences are made in the cell.Hence, the nucleic acid sequence of SEQ ID NO.3 encodes the amino acidsequence of SEQ ID NO.1. The nucleic acid sequence of SEQ ID NO.4encodes the amino acid sequence of SEQ ID NO.2. The amino acid sequencesof SEQ ID NO. 1 and SEQ ID NO.2 as they are formed in the cell eachcomprise a leader sequence. The leader sequence of amino acid sequenceSEQ ID NO.1 is from amino acid Nr. 1-20 of SEQ ID NO.1. The leadersequence of amino acid sequence SEQ ID NO.2 is from amino acid Nr. 1-19of SEQ ID NO.2. The leader sequence may directs the amino acid chain tothe surface of the cell. The leader sequence can be cleaved from thenascent amino acid chain and is not present in the finished protein. Theconstant domain of an engineered γ9δ2T-cell receptor may have shortconnecting sequences in which cysteine residues form a disulfide bond,forming a link between the γ9-TCR chain and the δ2-TCR chain. Forexample, C263 of amino acid sequence SEQ ID NO.1 and C249 of amino acidsequence SEQ ID NO.2, corresponding to the γ9-TCR chain and the δ2-TCRchain of G115wt may form a disulphide bond. A disulfide bond is acovalent bond formed by the coupling of two thiol groups of thecysteins.

In another embodiment, a nucleic acid sequence encoding the γ9δ2T-cellreceptor, or fragment thereof, according to the invention is provided,as well as a nucleic acid sequence comprising genetic constructs or agenetic construct encoding the γ9δ2T-cell receptor according to theinvention. It is understood that in this embodiment the nucleic acidsequences encoding the γ9δ2T-cell receptor are to be comprised in singlenucleic acid sequence. Preferably, the nucleic acid sequence comprisingthe genetic construct or genetic constructs is comprised in a retroviralvector.

In one embodiment, a cell is provided expressing the soluble fragment ofthe γ9δ2T-cell receptor according to the invention.

In one embodiment, a T cell is provided comprising the γ9δ2T-cellreceptor according the invention, i.e. corresponding to the specificγ9δ2T-cell receptors of which the CDR3 regions are substituted with thespecific amino acid sequences such as listed and indicated above. In afurther embodiment, the T cell comprises the isolated nucleic acidsequences as listed above, the genetic construct or genetic constructsaccording as listed above or the retroviral vector or retroviral vectorsas listed above.

Furthermore, the γ9δ2T-cell receptor, or fragment thereof, according tothe invention as described above, or a conjugate, an isolated nucleicacid sequence, or a genetic construct, or a retroviral vector, or a Tcell, as listed above, is for use as a medicament. Preferably, theγ9δ2T-cell receptor, or fragment thereof, according to the invention asdescribed above, or a conjugate, an isolated nucleic acid sequence, or agenetic construct, or a retroviral vector, or a T cell, as listed above,is for use as a medicament in the treatment of cancer.

It is understood that the specific γ9δ2T-cell receptors, or fragmentsthereof, according the invention, i.e. corresponding to the specificγ9δ2T-cell receptors of which the CDR3 regions are substituted with thespecific amino acid sequences such as listed and indicated above, are inparticular useful in medical treatments and/or in diagnostics. Forexample, immune cells may be redirected against cancer cells by e.g. exvivo transfer of one of the specific γ9δ2T-cell receptors as listed intoαβT-cells of a patient followed by expansion and adoptive transfer ofthese engineered T-cells back into the patient. Hence, immune cells maybe redirected against cancer cells. This may also be done in incombination with any other receptors (e.g. NKG2D) in immune-cells inorder to increase anti-cancer reactivity.

EXAMPLES Materials and Methods

Cells and Cell Lines

PBMCs were isolated from buffy coats obtained from Sanquin Blood Bank(Amsterdam, The Netherlands). Primary AML blasts were received afterobtaining informed consent from the LML biobank UMC Utrecht and a kindgift from Matthias Theobald (Mainz, Germany) and were collectedaccording to GCP and Helsinki regulations. Cell lines are described insupplementary Material and Methods.

TCR Mutagenesis, Cloning and Sequencing

γ9δ2TCR modifications are based on codon-optimized genes of γ9- orδ2-TCR chain G115 flanked by NcoI and BamHI restriction sites(synthesized by GeneArt, Regensburg, Germany). To generatealanine-mutations, site-directed mutagenesis was performed by overlapextension PCR 21 or whole plasmid mutagenesis 22; 23, using aproofreading polymerase (Phusion, Bioke). Mutated NcoI-BamHI digestedγ9- or δ2-TCR chains were ligated into the retroviral vector pBullet andsequenced by BaseClear (Leiden, The Netherlands).

Flow Cytometry

γ9δ2TCR expression was analyzed by flow cytometry using a Vδ2-FITC(clone B6, BD) or a pan-γδTCR-PE antibody (clone IMMU510, BeckmanCoulter). Fold change was calculated based on MFI values ofγ9-G115wt/δ2-G115wt transduced T-cells set to 1 and mock transducedT-cells to 0.

Functional T-Cell Assays

51Chromium-release assay for cell-mediated cytotoxicity was previouslydescribed. Target cells were labeled overnight with 100 μCu 51Cr (150μCu for primary cells) and incubated for 5 h with transduced T-cells infive effector-to-target ratios (E:T) between 30:1 and 0.3:1. Fold changewas calculated when compared to reactivity of engineered T-cellsexpressing unmutated γ9δ2TCR. IFN-γ ELISpot was performed usinganti-hulFN-ymAb1-D1K (I) and mAb7-B6-1 (II) (Mabtech-Hamburg, Germany)following the manufacturer's recommended procedure. Target and effectorcells (E:T 3:1) were incubated for 24 h in the presence of pamidronate(Calbiochem, Germany) where indicated. IFNγ ELISA was performed usingELISA-ready-go! Kit (eBioscience) following manufacturer's instructions.Effector and target cells (E:T 1:1) were incubated for 24 h in thepresence of pamidronate as indicated. Where specified, fold change wascalculated when compared to reactivity of engineered T-cells expressingunmutated γ9δ2TCR.

Retroviral Transduction of T-Cells

γ9δ2TCRs were transduced into αβT-cells as previously described(Marcu-Malina et al., 2011). In brief, packaging cells (phoenix-ampho)were transfected with gag-pol (pHIT60), env (pCOLT-GALV) (Stanislawskiet al., 2001) and two retroviral constructs (pBullet) containing eitherγ9-chain-IRES-neomycine or δ2-chain-IRES-puromycine, using Fugene6reagent (Takara, Gennevilliers, France). Human PBMC activated with αCD3(30 ng/ml) (Orthoclone OKT3, Janssen-Cilag, Tilburg, The Netherlands)and IL2 (50 IU/ml) (Proleukin, Novartis, Arnhem, The Netherlands) weretwice transduced with viral supernatant within 48 hours in the presenceof 50 IU/ml IL-2 and 4 μg/ml polybrene (Sigma-Aldrich, Zwijndrecht, TheNetherlands). Transduced T-cells were expanded by stimulation withαCD3/CD28 Dynabeads (0.5×10⁶ beads/10⁶ cells) (Invitrogen) and IL-2 (50IU/ml) and selected with 800 μg/ml geneticin (Gibco, Karlsruhe, Germany)and 5 μg/ml puromycin (Sigma-Aldrich, Zwijndrecht, The Netherlands) forone week. Where indicated polyclonal CD4+ and CD8+TCR transduced T-cellswith were sorted based on CD4 or CD8 expression using CD4 and CD8 MACSseparating system (Miltenyi Biothech, Bergish Gladbach, Germany).Following selection TCR-transduced T-cells were expanded in vitro basedon a previously described REP protocol (Riddell and Greenberg, 1990).

Functional T-Cell Assays

⁵¹Chromium-release assay for cell-mediated cytotoxicity was previouslydescribed (Kuball et al., 2004). Target cells were labelled overnightwith 100 μCu ⁵¹Cr (150 μCu for primary cells) and subsequently incubatedwith transduced T-cells in five effector to target ratios (E:T) between30:1 and 0.3:1. After 4-6 h ⁵¹Cr-release was measured in thesupernatant. Fold change was calculated based on ⁵¹Cr-release ofγ9-G115wt/69-G115wt transduced T-cells normalized to 1; for side by sideexperiments based on γ9-3 wt/69-3 wt, γ9-5 wt/69-5 wt orγ9-G115wt/69-G115wt normalized to 1.

IFNγ ELISpot was performed using anti-hu IFN-γ mAb1-D1K (I) andmAb7-B6-1 (II) from

Mabtech (Hamburg, Germany) following manufactures' recommended procedure(Besold et al., 2007). In all assays target and effector cells (E:T 3:1)were incubated for 24 h in the presence of pamidronate (Calbiochem,Germany) where indicated.

IFN-γELISA was performed using ELISA-ready-go! Kit (eBioscienc)following manufacturers' instructions. Effector and target cells (E:T1:1) were incubated for 24 h in the presence of pamidronate asindicated. Where specified, fold change was calculated based on IFN-γsecretion γ9-G115wt/69-G115wt transduced T-cells normalized to 1; forthe side by side experiments based on γ9-3 wt/69-3 wt, γ9-5 wt/69-5 wtor γ9-G115wt/69-G115wt normalized to 1.

Animal Models

To induce tumour xenografts, sublethal total body irradiated (2Gy),11-17 weeks old RAG-2−/−/yc−/−−BALB/C mice were injected i.v. with0.5×10⁶ Daudi-Luc cells (a kind gift from Genmab Utrecht, TheNetherlands) or 5×10⁶ RPMI8226/S-Luc cells (Anton Martens, Utrecht, TheNetherlands) together with 10⁷ γ9δ2TCR+□ transduced T-cells. TheRAG-2^(−/−)/yc^(−/−)-BALB/C mice were originally obtained from AMCASb.v. (Amsterdam, the Netherlands). Mice were bred and housed in thespecific pathogen-free (SPF) breeding unit of the Central AnimalFacility of the University of Utrecht. All animal experiments wereconducted according to Institutional Guidelines after acquiringpermission from the local Ethical Committee for Animal Experimentation,and in accordance with current Dutch laws on Animal Experimentation. Allmice developed tumours, mainly growing in the bone marrow visualized invivo once a week by Biospace bioluminescent imaging. Mice wereanesthetized by isoflurane inhalation before they received anintraperitoneal injection of 100 μl of 25 mg/ml Beetle Luciferin(Promega, USA). Bioluminescence images were acquired using a thirdgeneration cooled GaAs intensified charge-coupled device camera,controlled by the Photo Vision software and analyzed with M³Visionsoftware (all from Photon Imager;

Biospace Laboratory). Mice received 0.6×10⁶ IU of IL2 (Proleukin®,Novartis) in IFA s.c. on day 1 (together with tumour cells) and every 21days till the end of the experiment. Pamidronate (10 mg/kg body weight)was applied in the indicated groups at day 1 i.v. and every 21 days i.p.Outgrowing tumours were visualized in vivo by Biospace bioluminescentimaging (BLI). Mice were anesthetized by isoflurane before they receivedan intraperitoneal injection (100 μl) of 25 mg/ml Beetle Luciferin(Promega). Bioluminescence images were acquired and analyzed withM3Vision software (Photon Imager, Biospace Laboratory).

Results

Anti-Tumour Reactivity of Individual γ9δ2T-Cell Clones

To investigate whether individual γ9δ2T-cell clones mediate differentialactivity against tumour cells compared to the parental γ9δ2T-cellpopulation, γ9δ2T-cells from a healthy donor were cloned by limitingdilution and tested against a broad panel of tumour cells in an IFNγELISpot. High variability in tumour recognition in terms of specificityand functional avidity was observed between individual γ9δ2T-cell clones(cl); compared to the original bulk population, cl5 and cl13 producedtwice as many IFNγ spots in response to Daudi and selectively generatedsignificant amounts IFNγ when challenged with K562, BT549 and MCF-7. Incontrast, cl3 and cl15 recognized solely Daudi cells. Surface expressionof γ9δ2TCR, NKG2D, CD158a, NKAT-2 and NKB-1 was examined.

Anti-Tumour Reactivity Mediated by Individual γ9δ2TCRs

To elucidate differences among γ9δ2TCRs of tumour-reactive clones,sequences of wildtype (wt) γ9- and δ2-TCR chains of cl3(γ9-cl3_(wt)/δ2-cl3_(wt)) and cl5 (γ9-cl5_(wt)/δ2-cl5_(wt)) weredetermined and aligned with γ9δ2TCR G115. All three γ9δ2TCRs differed intheir CDR3 domains: 1-3 amino acids between position γ109 and γ111 inγCDR3 and 4-8 amino acids between δ108 and δ112 in δCDR3. To determinewhether distinct γ9δ2TCRs mediate differential anti-tumour reactivity,individual γ9δ2TCR chains were cloned into the retroviral vector pBulletand linked to a selection marker as described. The wildtype-combinationsγ9-cl3_(wt)/δ2-cl3_(wt), γ9-cl5_(wt)/δ2-cl5_(wt) andγ9-G115_(wt)/δ2-G115_(wt) were transduced into peripheral bloodαβT-cells, selected by antibiotics and further expanded. γ9δ2TCR G115(γ9-G115_(wt)/δ2-G115_(wt)) served as control, as did cells transducedwith an empty vector cassette (mock). γ9δ2TCR-transduced T-cells showedsimilar γ9δ2TCR expression and were tested for their lytic activityagainst the tumour target Daudi in a ⁵¹Cr-release assay (FIG. 1A).T-cells expressing γ9-cl3_(wt)/δ2-cl3, had a 50 percent reduced abilityto lyse tumour cells (p<0.01), whereas T-cells withγ9-cl5_(wt)/δ2-cl5_(wt) were nearly twice as potent (p<0.01) as thecontrol γ9-G115_(wt)/δ2-G115_(wt). To determine whether the phenotypesof γ9δ2TCR-transduced cells with decreased or increased functionalavidity are also present on cytokine level a pamidronate-titration assaywas performed. Pamidronate treatment of Daudi cells blocks themevalonate-pathway downstream to IPP causing the accumulation of IPP andan enhanced cytokine secretion of responsive T-cells. To exclude NK-likeactivation CD4⁺ γ9δ2TCR-transduced T-cells, which lack the expression ofmajor NK-receptors like NKG2D, were selected by MACS-sorting.Transductants were tested at different concentrations of pamidronateagainst the tumour target Daudi. Mock-transduced T-cells which underwentequivalent stimulation but express an irrelevant αβTCR served ascontrol. IFNγ secretion was measured by ELISA and the half maximaleffective concentration (EC50) was calculated (FIG. 1B). In line withchanges observed for lytic capacity, T-cells transduced withγ9-cl3_(wt)/δ2-cl3_(wt) secreted lower amounts of IFNγ (max. 600 μg/ml),while T-cells expressing γ9-cl5_(wt)/δ2-cl5_(wt) produced higher levelsof IFNγ (max. 1300 pg/ml) at all pamidronate concentrations, relative tocontrol γ9-G115_(wt)/δ2-G115_(wt) (max. 800 pg/ml). Despite differentplateaus in IFNγ secretion, all selected mutants and the wildtypecontrol had a comparable pamidronate-EC50 (˜30 pg/ml). These resultsindicate that distinct γ9δ2TCR clones mediate different functionalavidity and the high variability among parental γ9δ2T-cell clones intumour recognition seems to be substantially regulated by the CDR3domains of individual γ9δ2T-cell receptors.

Combinatorial-γδTCR-chain-exchange (CTE) as rapid method to modulatefunctional avidity of engineered T-cells.

To make the above determination, we devised a strategy namedcombinatorial-γδTCR-chain-exchange (CTE), which results in theexpression of newly combined γ9- and δ2-TCR chains on engineeredT-cells. During this process, γ9-G115_(wt) was combined with δ2-cl3_(wt)or δ2-cl_(wt) and δ2-G115_(wt) with γ9-cl3_(wt) or γ9-cl5_(wt). Thesecombinations were retrovirally transduced into αβT-cells. In alltransductants equivalent γδTCR expression was detected while theendogenous αβTCR was clearly down regulated. This resulted not only intoa nearly abolished allo-reactivity of αβT-cells expressingγ9-G115_(wt)/δ2-G115_(wt) but also of selected CTE-engineered αβT-cellswhen compared to mock-transduced cells. Thus, reactivity ofCTE-engineered T-cells primarily depends on expressed OTCRs and not onresidual endogenous αβTCRs. Next, transductants were functionally testedagainst the tumour target Daudi in a ⁵¹Cr-release assay (FIG. 1C). Theexchange of γ9- or δ2-chains indeed caused notable differences. Comparedto the original TCR γ9-G115_(wt)/δ2-G115_(wt), the combination ofγ9-G115_(wt)/δ2-cl3_(wt), γ9-G115_(wt)/δ2-cl5_(wt) orγ9-cl5_(wt)/δ2-G115_(wt) mediated 40 to 70 percent increased specificlysis of tumour cells (all p<0.05). The same magnitude of recognitionwas observed when IFNγ production of CD4⁺ γδTCR-transduced T-cells wastested in a pamidronate titration assay (FIG. 1D). Moreover, only thecombination γ9-cl_(wt)/δ2-G115_(wt) led to decreased IFNγ production oftransduced cells at all pamidronate concentrations (max. 100 pg/ml),while all other CTE-γ9δ2 TCRs mediated an increased IFNγ-secretion (max.1000 pg/ml) as compared to control TCR γ9-G115_(wt)/δ2-G115_(wt) (max.800 pg/ml). Equal pamidronate-EC50s of ˜30 pg/ml were calculated for allresponsive γ9δ2 TCR-transduced cells.

To determine whether cell-cell interaction influences theresponse-kinetics differently than pamidronate stimulation, CTE-γ9δ2 TCRγ9-G115_(wt)/δ2-cl5_(wt) which mediates improved functional avidity andcontrol TCR γ9-G115_(wt)/δ2-G115_(wt) were tested in aneffecter-to-target ratio (E:T) titration assay (FIG. 1E), and an E:T-50was calculated. Interestingly, T-cells with γ9-G115_(wt)/δ2-cl5_(wt)responded differently with an E:T-50 of 0.3:1, compared to an E:T-50 of1:1 calculated for control cells expressing γ9-G115_(wt)/δ2-G115_(wt).To test whether the interaction between different TCRs and ligands—thusthe affinity—is indeed increased, cell-cell conjugates between Daudi andT-cells expressing either potentially high (γ9-G115_(wt)/δ2-cl5_(wt)) orlow (γ9-cl3_(wt)/δ2-G115_(wt)) affinity TCRs were measured by flowcytometry. Significantly more cell-cell interactions were observed whenγ9-G115_(wt)/δ2-cl5_(wt) was expressed as compared toγ9-cl3_(wt)/δ2-G115_(wt) and mock-transduced T-cells (FIG. 1F). Thiseffect did not depend on the presence of pamidronate.G115_(wt)/δ2-cl5_(wt) is therefore a high affinity γ9δ2TCR. Hence, CTEis an efficient method to rapidly engineer γ9δ2TCRs with increasedaffinity, mediating improved functional avidity in transduced T-cells.

Residues in δCDR3 and Jδ1 are involved in γ9δ2TCR stability and inmediating functional avidity of engineered αβT-cells

To elucidate the molecular requirements of δCDR3 to mediate optimalfunctional avidity, alanine-mutagenesis of a model δCDR3 (clone G115)was performed including the whole Jδ1 segment, as important residueshave also been reported within Jγ1. During an initial screening, fivesequence areas were found to either influence TCR expression orfunctional avidity of γ9δ2 TCR transduced T-cells. To clarify the degreeto which single residues are responsible for impaired γ9δ2 TCRexpression and lower TCR-mediated functional avidity, single alaninemutations were generated. The mutated and wildtype δ2-G115 chains wereexpressed in combination with γ9-G115_(wt) in αβT-cells and tested forγ9δ2TCR expression using a δ2-chain specific antibody (FIG. 2A). Threesingle alanine mutations caused a 70 percent lower TCR expression whencompared to the unmutated δ2-G115_(wt), namely δ2-G115_(L116A),δ2-G115_(F118A) and δ2-G115_(V124A) (Table 3). Comparable results wereobserved using antibodies directed against the γ9-chain or the constantdomain of the γδTCR, indicating the importance of δ2-G115_(L116),δ2-G115_(F118) and δ2-G115_(V124) for stable TCR expression. The crystalstructure of γ9δ2 TCR G115 supports our findings: δ2-G115_(L116),δ2-G115_(F118) and δ2-G115_(V124) are located in hydrophobic cores (FIG.2B) and could thus be crucial for the structural stability of the γ9δ2TCR G115.

To address the impact of single alanine mutations on functional avidity,a ⁵¹Cr-release assay was performed (FIG. 2C). Transductants with low TCRexpression (δ2-G115_(L116A), δ2-G115_(F118A) and δ2-G115_(V124A)) couldnot tumour cells effectively, as they demonstrated an 80 percent lowerlytic capacity when compared to cells transduced with δ2-G115_(wt).T-cells with mutation δ2-G115_(L109A) and δ2-G115_(I117A) (Table 3)properly expressed the TCR but showed a 70 percent reduced lyticactivity when compared to δ2-G115_(wt) expressing cells. Similar resultswere obtained when TCR mutants were transduced into CD4⁺ Jurkat cellsand IL-2 production was measured (data not shown). Reduction of lyticactivity was also seen when alanine substitutions δ2-G115_(L109A) andδ2-G115_(I117A) were introduced into the δ2-chain of γδTCR clone 3.These results indicate that not only residue δL109, but also δI117 inδCDR3 may be generally important for γ9δ2 TCRs to mediate functionalavidity (FIG. 2D). Sequence alignments between δ2-chains of clones 3, 5and G115 indicated that δL109 and δI117 may be conserved (Table 2).

TABLE 3 CDR3 sequence of γ9δ2TCR G115, clone 3 and clone 5

The table above shows an alignment of three clones and the numberinglisted is in accordance with IGMT. The γ9-G115_(wt) sequence listedcorresponds to amino acids C117-T142 of amino acid sequence SEQ IDNO.1.γ112.1L, of either clone 3, 5 or G115_(wt) thus corresponds to L126of amino acid sequence SEQ ID NO.1. The δ2-G115wt sequence listedcorresponds to amino acids C111-C138 of amino acid sequence SEQ ID NO.2.Hence, as this alignment shows, corresponding CDR3 regions of clones 3and 5 can easily be identified via alignment.

Influence of CDR3 Length on Functional Avidity of γ9δ2TCR TransducedT-Cells

Alanine substitutions during alanine-scanning mutagenesis of γ9δ2 TCRG115 could replace large parts of the 8CDR3 domain without functionalconsequences. That raises the possibility that the crucial factor forthe differing functional avidities of distinct γ9δ2 TCR combinations mayalso involve the relative length between the functionally importantresidues δ2-G115₁₋₁₀₉ and the structurally important residueδ2-G115_(L116). Therefore, different δ2-G115 length mutants weregenerated. Since the triple δ2-G115_(T113-K115) is also important forstable surface expression (data not shown), nine length mutants(δ2-G115_(LM)) with 0 to 12 alanine between δ2-G115_(L109) andδ2-G115_(T113) were generated and equally expressed in αβT-cells, againin combination with γ9-G115_(wt) (FIG. 3A). To test the functionalavidity of δ2-G115_(LM) transduced T-cells, CD4⁺ TCR-transduced T-cellswere selected by MACS-sorting and an IFNγ ELISA in response to Daudi wasperformed in the presence of pamidronate (FIG. 3B). Engineered T-cellsexpressing δ2-G115_(LM0) and δ2-G115_(LM1) were unable to produce IFNγand T-cells expressing δ-G115_(LM4) or δ-G115_(LM12) secreted only abouthalf the amount of IFNγ compared to δ2-G115_(wt) transduced cells. Allother mutants (δ2-G115_(LM2, 3, 5, 6, 9)) induced comparable amounts ofIFNγ in engineered T-cells relative to transductants expressingδ2-G115_(wt). Mutants with functional impairment (δ2-G115_(LM0,1,4,12),Table 4) were further tested against increasing pamidronateconcentrations and an EC50 was calculated. Despite different plateaus inmaximal IFNγ secretion, all selected δ2-G115_(LM) transduced cells andthe wildtype control had a comparable pamidronate-EC50 (˜30 pg/ml) (FIG.3C). Length mutations were also studied in γCDR3 of γ9δ2 TCR G115 byengineering stretches of 1-6 alanines between γ9-G115_(E108) andγ9-G115_(E111.1) (γ9-G115₁₋₆). However, this did not affect functionalavidity.

This indicates that considerable alanine stretches within γ9 and δ2CDR3domains can be tolerated. However, too short and very long alaninestretches between δ2-G115_(L109) and δ2-G115_(T113) in particular, aswell as stretches with four alanines, may be associated with reduced orabsent function of a γ9δ2 TCR (FIGS. 3B and 3C).

Consequences for the Physiological γ9δ2 T-Cell Repertoire

The ImMunoGeneTics (IMGT) database was searched for reported stretchesbetween γ9-G115_(E109) and γ9-G115_(E111.1) as well as δ2-G115_(L109)and δ2-G115_(T113). A preferential length for reported γ9-chains wasfound for CDR3 regions corresponding to γ9-G115_(LM2) and γ9-G115_(LM3),but shorter stretches were also reported. In contrast, δ2-chains withshort δCDR3 domains, such as δ2-G115_(LM1) or δ2-G115_(LM0), were notreported (FIG. 3D), in line with our observation that such chains maynot be functional. The majority of listed γ9δ2TCRs contain δCDR3 lengthswhich correspond to δ2-G115_(LM5, 6,7). These findings support apreference to select γ9δ2TCRs with a δCDR3 length of 5-7 residuesbetween δ2-G115_(L109) and δ2-G115_(T113). Nevertheless, individualsequence differences can still play a role in γ9δ2TCR mediatedfunctional avidity.

Influence of the CDR3 Sequence on γ9δ2TCR Mediated Functional Avidity.

To test both the length and sequence of δCDR3 for mediating optimalfunctional avidity, γ9δ2TCR length mutants δ2-G115_(LM2), δ2-G115_(LM4),and δ2-G115_(LM6) were transduced into αβT-cells in combination withγ9-G115_(wt). IFNγ-secretion of transductants in response to Daudi wascompared to cells transduced with wildtype sequences from δ2-cl3_(wt)(corresponds in length to δ2-G115_(LM2)) δ2-cl5_(wt) (corresponds inlength to δ2-G115_(LM4)), and δ2-G115_(wt) (corresponds in length toδ2-G115_(LM6)) (Table 3). T-cells transduced with δ2-G115_(LM6) andδ2-G115_(wt) did not differ in the amount of cytokine secretion, allother combinations of wildtype chains showed a more than two-foldincrease in IFNγ when compared to the length mutant that selectivelycontained alanines (FIG. 3E). These results were confirmed when thelytic capacity of transduced cells was tested. The sequence in 8CDR3 maytherefore also be a significant factor for the functioning of a γ9δ2TCR.

Accordingly, the sequential importance of γCDR3 was studied. Thereby,γ9-G115_(LM1-3) were transduced into T-cells in combination withδ2-G115_(wt). IFNγ-secretion of transductants in response to Daudi wascompared to cells transduced with γ9-cl3_(wt) (corresponds in length toγ9-G115_(LM1)), γ9-cl5_(wt) (corresponds in length to γ9-G115_(LM2)) andγ9-G115_(wt) (corresponding to γ9-G115_(LM3)) (Table 3). T-cellsexpressing γ9-cl3_(wt)/82-G115_(wt) selectively produced lower amountsof IFNγ when compared to their equivalent γ9-G115_(LM1) (FIG. 4A).Previously, the same γ9δ2TCR combination was also found to mediatereduced functional avidity (FIGS. 1C and 1D). Loss of activity could berestored to normal levels (referred to γ9δ2TCR G115_(wt)) by mutatingγCDR3_(E109) in γ9-cl3_(wt) to γCDR3_(A109), demonstrating that a singlechange in the variable sequence of γ9CDR3 may be sufficient to regulatefunctional avidity of γ9δ2TCR transduced T-cells tested here.

In summary, the length and sequence of the δ2CDR3 domain between L109and T113 (Table 3) can play a role in γ9δ2 TCR-mediated functionalavidity. In addition, the individual sequence between E108 and E111.1 inγ9CDR3 may hamper activity of a γ9δ2TCR, and in G115 γCDR3_(A109) may beof importance for ligand interaction (Table 3 and FIG. 4B). Combined,this provides a rationale for CTE-engineered γ9δ2TCRs but also forrandom mutagenesis within both γ9 and δ2CDR3 regions.

CTE-Engineered T-Cells as a Tool for Cancer Immunotherapy.

CTE-engineered γ9δ2 TCRs with increased activity against tumour cellsare interesting candidates for TCR-gene therapeutic strategies. Changesin functional avidity mediated by CTE-γ9δ2 TCRs may constitute a uniquephenomenon of a defined γ9δ2 TCR pair in response to the B-lymphoblasticcell line Daudi, or this may be a general response to most tumourtargets. Therefore, CTE-γ9δ2 TCRs that mediated increased(γ9-G115_(wt)/δ2-cl5_(wt)) or reduced (γ9-cl3_(wt)/δ2-G115_(wt))activity were tested against various tumours in an IFNγELISA in thepresence of pharmacological concentrations of pamidronate (10 μM) (FIG.5A). Tumour reactivity was significantly increased against a whole rangeof different tumour entities including other hematological cancers suchas RPMI8226/S, OPM2, LME1 (all multiple myeloma), K562 (myelogenousleukemia) as well as solid cancer cell lines such as Saos2(osteosarcoma), MZ1851RC (renal cell carcinoma), SCC9, Fadu (head andneck cancer), MDA-MB231, MCF7, BT549 (all breast cancer), and SW480(colon carcinoma) when taking advantage of γ9-G115_(wt)/δ2-cl5_(wt) ascompared to γ9-G115_(wt)/δ2-G115_(wt) and was significantly reduced oreven absent for all other targets using γ9-cl3_(wt)/δ2-G115_(wt).Moreover, CTE-engineered T-cells with increased activity against tumourcells still did not show any reactivity towards healthy tissue such asPBMCs and fibroblasts. Superior lytic activity of T-cells engineeredwith γ9-G115_(wt)/δ2-cl5_(wt) was also observed for hematological cancercells like RPMI8226/S, OPM2, L363 as well as solid cancer cell linesSaos2, MZ1851RC, SCC9, MDA-MB231, and SW480 when compared to controlT-cells expressing γ9-G115_(wt)/δ2-G115_(wt) (FIG. 5B). Therefore,CTE-engineered γ9δ2 TCRs can provide higher anti-tumour response againsta broad panel of tumour cells while not affecting normal tissue, andthus have the potential to increase efficacy of TCR-engineered T-cells.

To assess the potential clinical impact of CTE-engineered γ9δ2 TCRs, itwas tested whether an increased efficacy of CTE-γ9δ2 TCRs may also bepresent when primary blasts of AML patients are chosen as targets.Therefore, CTE-γ9δ2 TCR transduced T-cells were tested against 11primary AML blasts and healthy CD34⁺ progenitor cells in an IFNγ-ELISpot(FIG. 5C). Transductants expressing γ9-G115_(wt)/δ2-cl5_(wt) recognized8 out of 11 primary AML samples equally or superiorly compared tocontrol γ9-G115_(wt)/δ2-G115_(wt). Furthermore, CD34⁺ progenitor cellswere not recognized by T-cells expressing eitherγ9-G115_(wt)/δ2-cl5_(wt) or γ9-G115_(wt)/δ2-G115_(wt). In light of thesefindings, CTE-engineered TCR γ9-G115_(wt)/δ2-cl5_(wt) appears to be apromising candidate for clinical application.

Finally, to demonstrate that CTE-γ9δ2 TCRs are safe and function withincreased efficacy when compared to the original constructs in vivo,adoptive transfer of T-cells engineered with CTE-TCRs was studied in ahumanized mouse model: protection against outgrowth of Daudi orRPMI8226/S in Rag2^(−/−)yc^(−/−) double knockout mice. Thereforeperipheral blood αβT-cells were transduced with CTE-TCRγ9-G115_(wt)/δ2-cl5_(wt) or control TCR γ9-G115_(wt)/δ2-G115_(wt).CTE-TCR transduced T-cells showed similar expression of homing markersincluding L-selectin and CCR7. Irradiated Rag2^(−/−)yc^(−/−) micereceived luciferase-transduced Daudi (0.5×10⁶) or RPMI8226/S cells(5×10⁶) and 10⁷ CTE-engineered T-cells by intravenous injection. Thefrequency of T-cell infusion was reduced to one intravenous injectionrelative to our previously reported model were two infusions were givenin order to test superiority of CTE-TCR transduced T-cells undersuboptimal conditions. Consequently, this resulted in loss of protectionwith TCR G115_(wt)-engineered T-cells when tumour growth was measured bybioluminescence imaging (BLI) (FIGS. 6A and 6B). However, CTE-engineeredT-cells expressing γ9-G115_(wt)/δ2-cl5_(wt) clearly reduced tumouroutgrowth for Daudi (20.000 counts/min, day 42, n=4) and RPMI8226/S(80.000 counts/min, day 35, n=7) as compared to TCR G115_(wt)-engineeredT-cells (Daudi: 180.000 counts/min, day 42; RPMI8226/S: 210.000counts/min, day 35). T-cells could be found in the periphery until 1-2weeks after infusion in mice, but frequency of T-cells did not correlatewith tumour regression. Finally, in the rapidly lethal Daudi-model onlymice treated with CTE-engineered T-cells had a significant increasedoverall survival of ˜2 months relative to mice treated with T-cellsexpressing γ9-G115_(wt)/δ2-G115_(wt) (FIG. 6C). These results indicatethat CTE-engineered γ9δ2 TCRs efficiently mediate anti-tumour reactivityin vivo, which points to CTE as a potential tool to optimize γ9δ2TCRsfor clinical application.

In table 4 below, results with regard to methods performed according tothe invention are listed. The delta-CDR3 domain was tested for TCRfunction by alanine-mutagenesis (Table 3, mutation) of the CDR3-domain(including the whole J-segment) of the well studied clone G115 and aminoacids that may be relevant for TCR stability and TCR function are found.In addition, the amino acid sequence length (Table 3, length mutations)between γE108 and γE111.1 of the γ9-chain and between δL109 and 5T113 ofthe δ2-chain was tested. The individual sequences of γ9-CDR3 and δ2-CDR3domains are of importance for function (Table 3, sequence). ByCTE-engineering the γ9- and δ2-TCR chains of clone G115 was combinedwith γ9- and δ2-TCR chains of clone 3 and clone 5, respectively.CTE-engineering resulted in 4 newly designed γδTCRs. The original TCRsand the new CTE-engineered TCRs (Table 3, combination) were transducedinto αβT-cells and tested for their function

TABLE 4 Effect of mutations and chain-combinations on γ9δ2-TCR functionIFN-γ cytotoxic γ9δ2TCR expression response activity mutationγ9-G115/δ2-G115 original +++ +++ +++ γ9-G115/δ2-G115_(T108A)  δT108 →δA108 ++++ +++ +++ γ9-G115/δ2-G115_(L109A)  δL109 → δA109 ++++ − −γ9-G115/δ2-G115_(G110A) δG110 → δA110 +++ nd +++ γ9-G115/δ2- δM111 →δA111  +++ +++ +++ G115_(M111A, G111.1A, G112.2A) δG111.1 → δA111.1δG112.2 → δA112.2 γ9-G115/δ2-  δE112.1 → δA112.1 +++ +++ +++G115_(E112.1A, Y112A, T113A) δY112 → δA112  δT113 → δA113γ9-G115/δ2-G115_(D114A) δD114 → δA110 +++ nd +++ γ9-G115/δ2-G115_(K115A)δK115 → δA110 +++ nd +++ γ9-G115/δ2-G115_(L116A)  δL116 → δA116 − nd −γ9-G115/δ2-G115_(I117A)  δI117 → δA117 +++ − − γ9-G115/δ2-G115_(F118A) δF118 → δA118 − nd − γ9-G115/δ2-G115_(G119A) δG119 → δA119 +++ nd +++γ9-G115/δ2- δK120 → δA120 +++ +++ +++ G115_(K120A, G121A, T122A) δG121 →δA121  δT122 → δA122 γ9-G115/δ2-G115_(R123A) δR123 → δA123 +++ nd +++γ9-G115/δ2-G115_(V124A) δV124 → δA124 − nd − γ9-G115/δ2-G115_(T125A) δT125 → δA125 +++ nd +++ γ9-G115/δ2-G115_(V126A) δV126 → δA126 +++ nd+++ γ9-G115/δ2-G115_(E127A)  δE127 → δA127 +++ nd +++γ9-G115/δ2-G115_(P128A)  δP128 → δA128 +++ nd +++ γ9-G115/δ2- deletionδT113, + nd nd G115_(deletion T113-K115) δD114 & δK115 length mutationγ9-G115_(LM1)/δ2-G115 γE108-1A-γE111.1 +++ +++ +++ γ9-G115_(LM2)/δ2-G115γE108-2A-γE111.1 +++ +++ +++ γ9-G115_(LM3)/δ2-G115 γE108-3A-γE111.1 ++++++ +++ γ9-G115_(LM4)/δ2-G115 γE108-4A-γE111.1 +++ +++ +++γ9-G115_(LM5)/δ2-G115 γE108-5A-γE111.1 +++ +++ +++ γ9-G115_(LM6)/δ2-G115γE108-6A-γE111.1 +++ +++ +++ γ9-G115/δ2-G115_(LM0) δL109-0A-δT113 +++ −− γ9-G115/δ2-G115_(LM1) δL109-1A-δT113 +++ − − γ9-G115/δ2-G115_(LM2)δL109-2A-δT113 +++ +++ +++ γ9-G115/δ2-G115_(LM3) δL109-3A-δT113 +++ ++++++ γ9-G115/δ2-G115_(LM4) δL109-4A-δT113 +++ + +++ γ9-G115/δ2-G115_(LM5)δL109-5A-δT113 +++ +++ +++ γ9-G115/δ2-G115_(LM6) δL109-6A-δT113 +++ ++++++ γ9-G115/δ2-G115_(LM9) δL109-9A-δT113 +++ ++ +++γ9-G115/δ2-G115_(LM12) δL109-12A-δT113 +++ + +++ sequence γ9-G115/δ2-cl3original +++ +++ +++ γ9-G115/δ2-G115_(LM2) δL109-2A-δT113 +++ + +γ9-G115/δ2-cl5 original +++ +++ +++ γ9-G115/δ2-G115_(LM4) δL109-4A-δT113+++ + + γ9-cl3/δ2-G115 original +++ nd + γ9-cl3_(E109A)/δ2-G115  γE109 →γA109 +++ nd +++ combination γ9-G115/δ2-G115 original +++ +++ +++γ9-cl3/δ2-cl3 original +++ + + γ9-cl5/δ2-cl5 original +++ +++++ +++++γ9-cl3/δ2-G115 CTE +++ + + γ9-G115/δ2-cl3 CTE +++ ++++ ++++γ9-cl5/δ2-G115 CTE +++ ++++ ++++ γ9-G115/δ2-cl5 CTE +++ +++++ +++++

SEQ LISTING SEQ ID description type 1 γ9-T-cell receptor chain (G115) AA2 δ2-T-cell receptor chain (G115) AA 3γ9-T-cell receptor chain CDR3 region AA (G115) 4δ2-T-cell receptor chain CDR3 region  AA (G115) 5γ9-T-cell receptor chain (G115) NA 6 δ2-T-cell receptor chain (G115) NA7 γ9-T-cell receptor chain (clone 3) NA 8γ9-T-cell receptor chain (clone 3) AA 9δ2-T-cell receptor chain (clone 3) NA 10δ2-T-cell receptor chain (clone 3) AA 11γ9-T-cell receptor chain (clone 5) NA 12γ9-T-cell receptor chain (clone 5) AA 13δ2-T-cell receptor chain (clone 5) NA 14δ2-T-cell receptor chain (clone 5) AA 15 ALKRTD AA 16 ALWEIQELGKKIKV AA17 ACDALKRTDTDKLI AA 18 ACDLLGYTDKLI AA 19 TLGMGGEY AA AA = amino acidsequence NA = nucleic acid sequence

1. A method for identifying γ9δ2T-cell receptors that mediateanti-tumour responses comprising the steps of: a) providing T-cells; b)providing a nucleic acid sequence encoding a γ9-T-cell receptor chaincomprising a γ9-CDR3 encoding region, wherein the γ9-CDR3 encodingregion is randomly modified, and a nucleic acid sequence encoding aδ2-T-cell receptor chain comprising a δ2-CDR3 encoding region, whereinthe δ2-CDR3 encoding region is randomly modified; c) introducing thenucleic acid sequences of step b) into the T-cells to provide for anengineered T-cell with a γ9δ2T-cell receptor comprising the γ9-T-cellreceptor chain of step b) and the δ2-T-cell receptor chain of step b);d) optionally, repeating steps b) and c); e) determining the anti-tumourresponses of the engineered T-cells provided in steps c) and d); f)identifying the γ9δ2T-cell receptors of the engineered T-cells thatmediate anti-tumour responses.
 2. The method according to claim 1,wherein the nucleic acid sequence encoding a γ9-T-cell receptor chainencodes an amino acid sequence having at least 60% sequence identitywith the amino acid sequence of SEQ ID NO. 1, and/or wherein the nucleicacid sequence encoding a δ2-T-cell receptor chain encodes an amino acidsequence having at least 60% sequence identity with the amino acidsequence of SEQ ID NO.
 2. 3. The method according to claim 1, whereinthe randomly modified γ9-CDR3 encoding region is modified at the aminoacid sequence corresponding to amino acid residues 122-124 of SEQ IDNO
 1. 4. The method according to claim 1, wherein the randomly modifiedδ2-CDR3 encoding region is modified at the amino acid sequencecorresponding to amino acid residues 115-122 of SEQ ID NO.
 2. 5. Themethod according to claim 4, wherein randomly modifying the amino acidsequence corresponding to the δ2-CDR3 encoding region comprises amodification in length.
 6. The method according to claim 1, whereinrandomly modifying the amino acid sequence comprises introducing aminoacid substitutions, deletions, and/or insertions.
 7. The methodaccording to claim 1, wherein the nucleic acid sequence encoding aγ9-T-cell receptor chain is provided in an expression vector and thenucleic acid sequence encoding a δ2-T-cell receptor chain is provided inan expression vector, or wherein the nucleic acid sequence encoding aγ9-T-cell receptor chain and the nucleic acid sequence encoding aδ2-T-cell receptor chain are provided in a single expression vector. 8.The method according to claim 1, wherein the nucleic acid sequenceencoding a γ9-T-cell receptor chain is provided in a retroviral vectorand the nucleic acid sequence encoding a δ2-T-cell receptor chain isprovided in a retroviral vector, or wherein the nucleic acid sequenceencoding a γ9-T-cell receptor chain and the nucleic acid sequenceencoding a δ2-T-cell receptor chain are provided in a single retroviralvector, and wherein the steps of introducing the nucleic acid sequencesinto the T-cells comprises retroviral vector transduction of theT-cells.
 9. The method according to claim 1, wherein the step ofdetermining the anti-tumour responses comprises contacting theengineered T-cell with a tumour cell and measuring its ability to lysethe tumour cell and/or induce IFN-γ.
 10. A γ9δ2T-cell receptor, or afragment thereof, comprising a γ9-T-cell receptor chain comprising aγ9-CDR3 region, wherein the amino acid sequence of the γ9-CDR3 regioncorresponding to amino acid residues 122-124 of SEQ ID NO. 1 is modifiedand a δ2-T-cell receptor chain comprising a δ2-CDR3 region wherein theamino acid sequence of the δ2-CDR3 region corresponding to amino acidresidues 115-122 of SEQ ID NO. 2 is modified, wherein the combinationsof the amino acid modifications within the γ9δ2T-cell receptor, or afragment thereof, are as listed in Table
 1. 11. A γ9δ2T-cell receptor,or a fragment thereof, comprising: a γ9-T-cell receptor chain comprisinga γ9-CDR3 region, wherein the amino acid sequence of the γ9-CDR3 regioncorresponding to amino acid residues 122-124 of the amino acid sequenceof SEQ ID NO. 1 is AQQ, and a δ2-T-cell receptor chain comprising aδ2-CDR3 region wherein the amino acid sequence of the δ2-CDR3 regioncorresponding to amino acid residues 115-122 of the amino acid sequenceof SEQ ID NO. 2 is ALKRTD.
 12. The γ9δ2T-cell receptor or fragmentthereof according to claim 10, wherein the amino acid sequence of theγ9-CDR3 region corresponding to amino acid residues 118-121 of the aminoacid sequence of SEQ ID NO. 1 is ALWE, and wherein the amino acidsequence of the γ9-CDR3 region corresponding to amino acid residues125-132 of the amino acid sequence of SEQ ID NO. 1 is ELGKKIKV, andwherein the amino acid sequence of the δ2-CDR3 region corresponding toamino acid residues 112-114 of the amino acid sequence of SEQ ID NO. 2is ACD, and wherein the amino acid sequence of the δ2-CDR3 regioncorresponding to amino acid residues 123-127 of the amino acid sequenceof SEQ ID NO. 2 is TDKLI.
 13. The γ9δ2T-cell receptor, or fragmentthereof, according to claim 10, wherein the γ9-T-cell receptor chaincomprises an amino acid sequence having at least 60% sequence identitywith the amino acid sequence of SEQ ID NO. 1, and wherein the δ2-T-cellreceptor chain comprises an amino acid sequence having at least 60%sequence identity with the amino acid sequence of SEQ ID NO.
 2. 14. Theγ9δ2T-cell receptor, or fragment thereof, according to claim 10, whereinthe γ9-T-cell receptor chain comprises an amino acid sequence comprisingamino acid residues 1-121 and 125-315 of the amino acid sequence of SEQID NO. 1 and wherein the δ2-T-cell receptor chain comprises an aminoacid sequence comprising amino acid residues 1-114 and 123-293 of theamino acid sequence of SEQ ID NO. 2 and.
 15. A conjugate comprising asoluble fragment of the γ9δ2T-cell receptor according to claim 10 linkedto an agent.
 16. The conjugate according to claim 15, wherein the agentis selected from the group consisting of a diagnostic agent, atherapeutic agent, an anti-cancer agent, a chemical, a nanoparticle, achemotherapeutic agent and a fluorochrome.
 17. An isolated nucleic acidsequence encoding the δ2-T-cell receptor chain, or fragment thereof, asdefined in claim 10, wherein the δ2-CDR3 region corresponding to aminoacid residues 115-122 of the amino acid sequence of SEQ ID NO. 2 issubstituted with amino acid residues ALKRTD or LLGY.
 18. An isolatednucleic acid sequence encoding the γ9-T-cell receptor chain, or fragmentthereof, as defined in claim 10, wherein the amino acid sequence of theγ9-CDR3 region corresponding to amino acid residues 122-124 of the aminoacid sequence of SEQ ID NO. 1 is substituted with amino acid residuesIQ.
 19. A genetic construct comprising the isolated nucleic acidsequence according to claim
 17. 20. A retroviral vector comprising agenetic construct according to claim
 19. 21. An isolated nucleic acidsequence encoding the γ9δ2T-cell receptor, or fragment thereof, asdefined in claim
 10. 22. A genetic construct comprising the nucleic acidsequence according to claim
 21. 23. A retroviral vector comprising thegenetic construct according to claim
 22. 24. A cell expressing a solublefragment of the γ9δ2T-cell receptor as defined in claim
 15. 25. A T cellcomprising the γ9δ2T-cell receptor according to claim
 10. 26. A T cellaccording to claim 25, wherein the T cell comprises: a) an isolatednucleic acid sequence encoding: i) a δ2-T-cell receptor chain, orfragment thereof, comprising a δ2-CDR3 region, wherein the amino acidsequence of the δ2-CDR3 region corresponding to amino acid residues115-122 of the amino acid sequence of SEQ ID NO. 2 is substituted withamino acid residues ALKRTD or LLGY, or ii) an isolated nucleic acidsequence encoding a γ9-T-cell receptor chain, or fragment thereof,comprising a γ9-CDR3 region, wherein the amino acid sequence of theγ9-CDR3 region corresponding to amino acid residues 122-124 of SEQ IDNO. 1 is substituted with amino acid residues IQ; c) a genetic constructcomprising the isolated nucleic acid sequence or d) a retroviral vectorcomprising the genetic construct.
 27. A medicament comprising oneselected from the group consisting of: a) γ9δ2T-cell receptor, orfragment thereof, comprising: i) a γ9-T-cell receptor chain comprising aγ9-CDR3 region, wherein the amino acid sequence of the γ9-CDR3 regioncorresponding to amino acid residues 122-124 of SEQ ID NO. 1 is modifiedand a δ2-T-cell receptor chain comprising a δ2-CDR3 region wherein theamino acid sequence of the δ2-CDR3 region corresponding to amino acidresidues 115-122 of SEQ ID NO. 2 is modified, wherein the combinationsof the amino acid modifications within the γ9δ2T-cell receptor, or afragment thereof, are as listed in Table 1; or ii) a γ9-T-cell receptorchain comprising a γ9-CDR3 region, wherein the amino acid sequence ofthe γ9-CDR3 region corresponding to amino acid residues 122-124 of SEQID NO. 1 is AQQ and a δ2-T-cell receptor chain comprising a δ2-CDR3region wherein the amino acid sequence of the δ2-CDR3 regioncorresponding to amino acid residues 115-122 of SEQ ID NO. 2 is ALKRTD;b) a conjugate comprising a soluble fragment of the γ9δ2T-cell receptorlinked to an agent; c) an isolated nucleic acid sequence encoding: i)the δ2-T-cell receptor chain, or fragment thereof, wherein the δ2-CDR3region corresponding to amino acid residues 115-122 of SEQ ID NO. 2 issubstituted with amino acid residues ALKRTD or LLGY, ii) the γ9-T-cellreceptor chain, or fragment thereof, wherein the amino acid sequence ofthe γ9-CDR3 region corresponding to amino acid residues 122-124 of theamino acid sequence of SEQ ID NO. 1 is substituted with amino acidresidues IQ, or iii) the γ9δ2T-cell receptor, or fragment thereof; d) agenetic construct comprising the isolated nucleic acid sequence; e) aretroviral vector comprising the genetic construct; and f) a T cellcomprising the γ9δ2T-cell receptor.
 28. The medicament according toclaim 27, wherein the medicament is useful for the treatment of cancer.